Team:GeorgiaTech/WeekEight

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<style type="text/css">ol{margin:0;padding:0}p{margin:0}.c6{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c2{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c0{line-height:1.15;text-indent:0pt;direction:ltr}.c8{line-height:1.15;text-indent:36.0pt;direction:ltr}.c1{color:#ffffff;font-size:10pt;font-family:Arial}.c5{font-weight:bold}.c11{background-color:#ffffff}.c4{text-decoration:underline}.c9{list-style-type:decimal}.c10{text-decoration:line-through}.c3{list-style-type:disc}.c13{list-style-type:lower-latin}.c12{list-style-type:circle}.c7{font-style:italic}</style></head><body class="c11"><p class="c0"><span class="c1">Plan for this week:</span></p><p class="c0"><span class="c1">Weekend: PCR purify hybB, run gel for pSB1A3</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Mon: Redo PCR RFP; digest RFP (O/N)</span></li><li class="c2"><span class="c1">Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)</span></li><li class="c2"><span class="c1">Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N</span></li><li class="c2"><span class="c1">Thurs: Tranformation of cells by hyb+RFP+psb1a3</span></li><li class="c2"><span class="c1">Fri: check plates</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/20/2010</span></p><p class="c0"><span class="c1 c4">Goals</span><span class="c1">: </span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Redo PCR of mRFP F,R; mRFP F2,R</span></li><li class="c2"><span class="c1">Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes) </span></li><li class="c2"><span class="c1">RUN GEL - 2 uL each - to check size - this step completed for all building blocks</span></li><li class="c2"><span class="c1">PCR PURIFY leftovers - this step completed for all building blocks</span></li><li class="c2"><span class="c1">NANOSPEC - record concentrations - this step completed for all building blocks</span></li><li class="c2"><span class="c1">Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock</span></li></ol><p class="c0"><span class="c1 c4">Notes:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1 c5">Ryan gave us a fresh stock of mRFP plasmid</span><span class="c1">. </span><span class="c1 c4">We only get this one</span><span class="c1">! It is precious like gold or diamonds!</span></li><li class="c2"><span class="c1">Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest. </span></li></ol><p class="c0"><span class="c1 c4">mRFP</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">in the plasmid PET15B</span></li><li class="c2"><span class="c1">Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c5">9am </span></p><p class="c0"><span class="c1 c4">PCR Protocol </span><span class="c1"> for mRFP F,R; mRFP F2, R</span></p><p class="c0"><span class="c1">26.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c5">PHUSION 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1 c5">2 uL</span><span class="c1"> template DNA</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">PHUSION</span></p><p class="c0"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ran PCR results (pre purification) on gel)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">12pm</span></p><p class="c0"><span class="c1 c4">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c0"><span class="c1">10 &#1405;L Nova Blue cells + 5 &#1405;L of MRFP plasmid (from stock that Ryan gave us this morning).</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for Heat Shock Transformation </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results</span></p><ol class="c9"><li class="c2" value="1"><span class="c1">Gel picture of mRFP FR, F2R (Insert gel pic!)</span></li></ol><ol class="c13"><li class="c6" value="1"><span class="c1">The bands of both PCRs (pre digest) were between approximately around &nbsp;700-800 </span></li></ol><p class="c0"><img height="288.0" src="https://static.igem.org/mediawiki/2010/5/5d/9-19a.png" width="385.0"></p><p class="c0"><span class="c1 c7">Gel of mRFP FR and mRFP F2R</span></p><p class="c0"><span class="c1 c7">100bp ladder|mRFP FR|mRFP F2R|Control</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Purificatio</span><span class="c1">n-mRFP</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purification </span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec Results</span></p><p class="c0"><span class="c1">mRFP, F: 137.2ng/uL</span></p><p class="c0"><span class="c1">mRFP, F2: 132.3 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)</span></li><li class="c2"><span class="c1">Check if transformation of Nova Blue with mRFP worked!</span></li></ol><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/21/2010</span></p><p class="c0"><span class="c1">Results from 9/20/2010</span></p><p class="c0"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transformation of mRFP in NB cells successful (both plates). </span></p><p class="c0"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c0"><span class="c1 c4">Starter Cultures</span></p><p class="c0"><span class="c1">Make starter cultures from both mRFP:NB plates, allow to grow overnight. </span></p><p class="c8"><span class="c1">3mL LB + 3uL 1000X CARB</span></p><p class="c8"><span class="c1">put in 37C incubator with shaking overnight</span></p><p class="c8"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For 9/22/2010:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening</span></li><li class="c2"><span class="c1">Create cryostocks from starter cultures grown on 9/21/2010.</span></li><li class="c2"><span class="c1">Couldn&rsquo;t start RE digest of RFP today- could not find RE&rsquo;s. Will ask Megan Wednesday morning.</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/22/2010- (Mitesh, Scott, Gita)</span></p><p class="c0"><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Digest of RFP F,R</span></li><li class="c2"><span class="c1">PCR purify the digest</span></li><li class="c2"><span class="c1">Ligate hyBB FR, to RFP F,R</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes: made a stock of 1 ug/ul BSA for easier RE digests</span></p><p class="c0"><span class="c1">I am following the suggested RE protocol listed on the Promega literature that came with the RE&rsquo;s</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for RFP-F,R</span><span class="c1">r</span></p><p class="c0"><span class="c1">4.5 uL H20</span></p><p class="c0"><span class="c1">2uL 10X Promega Buffer D</span></p><p class="c0"><span class="c1">10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)</span></p><p class="c0"><span class="c1">2 uL BSA (0ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI (edited changed from notI to speI )</span></p><p class="c0"><span class="c1">0.75 uL NotI</span></p><p class="c0"><span class="c1 c5">Total=20 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">Start time is 10:35 am</span></p><p class="c0"><span class="c1">End time should be 1:35 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Purification of RFP digest</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purfication</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec</span></p><p class="c0"><span class="c1">RFP Digest (purified)- 41 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation of RFP-FR to HybB-FR</span></p><p class="c0"><span class="c1">using hybB from 9/18/2010 and RFP from 9/21/2010</span></p><p class="c0"><span class="c1 c7">Calculating equivalents:</span></p><p class="c0"><span class="c1">RFP- [41 ng/uL]/678 bp= 0.06 eq/uL</span></p><p class="c0"><span class="c1">hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL</span></p><p class="c0"><span class="c1">for linear ligations, use a 1:1 ratio of products</span></p><p class="c0"><span class="c1 c4">Ligation:</span></p><p class="c0"><span class="c1">2 uL of RFP FR (SpeI, NotI digested; purified)</span></p><p class="c0"><span class="c1">2 uL of HybB FR (EcoRI, NotI digested; purified)</span></p><p class="c0"><span class="c1">1 uL 10x Ligase Buffer</span></p><p class="c0"><span class="c1">4.5 uL H20</span></p><p class="c0"><span class="c1">0.5 uL T4 Ligase</span></p><p class="c0"><span class="c1 c5">Total=10 uL</span></p><p class="c0"><span class="c1">Leave RT for 1 hour</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Start time: 3:20 pm</span></p><p class="c0"><span class="c1">End time: 4:20 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR of hybB+Mrfp construct </span></p><p class="c0"><span class="c1">3 uL of ligation reaction</span></p><p class="c0"><span class="c1">25.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c5">PHUSION 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">PHUSION</span></p><p class="c0"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c0"><span class="c1">Start: 5pm</span></p><p class="c0"><span class="c1">End: Thursday</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Take out pcr of mrfp+hybB</span></li><li class="c2"><span class="c1">Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)</span></li><li class="c2"><span class="c1">Digest the construct, along with the vector psb1a3 at the same time (3 hrs)</span></li><li class="c2"><span class="c1">Ligate the construct to psb1a3 (1 hr)</span></li><li class="c2"><span class="c1">Transform into e coli (1.5 hrs)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9.23.2010 (Scott, Rob, Gita)</span></p><p class="c0"><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Run PCR of RFP+hyBb on gel</span></li><li class="c2"><span class="c1">Digest the construct, along with vecotr psb1a3</span></li><li class="c2"><span class="c1">ligate the construct to psb1a3</span></li><li class="c2"><span class="c1">transform into e. coli</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes:</span></p><p class="c0"><span class="c1">Predicted size of hyb+MRFP~1000bp</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Running PCR product on gel</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">The pcr was left overnight at 12 c, and I am running 4 uL on a gel</span></li><li class="c2"><span class="c1">4 &nbsp;uL sample + 1 uL running dye</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.</span></li><li class="c2"><span class="c1">PCR products store in yellow box</span></li><li class="c2"><span class="c1">Building blocks in blue rack</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR</span></p><p class="c0"><span class="c1">1. &nbsp;Heat 30 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">2. Add 35 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">3. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results</span></p><p class="c0"><img height="313.0" src="https://static.igem.org/mediawiki/2010/0/0f/9-19b.png" width="419.0"></p><p class="c0"><span class="c1 c7">Gel from 9.23.2010.</span></p><p class="c0"><span class="c1 c7">100 bp ladder| RFP-FR +HybB PCR|</span></p><p class="c0"><span class="c1 c7">Predicted Size- 1070 bp</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR (Hammer Lab Apparatus)</span></p><p class="c0"><span class="c1">1. &nbsp;Heat 90 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">2. Add 90 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">3. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">(I did 100 mL, but found the actual volume to be around 80 to 90 mL)</span></p><p class="c0"><span class="c1">Start: 4:10 pm</span></p><p class="c0"><span class="c1">End: 5:10 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Gel Extraction Protocol</span></p><p class="c0"><span class="c1">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c0"><span class="c1">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c0"><span class="c1">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c0"><span class="c1">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c0"><span class="c1">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c0"><span class="c1">6. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.</span></p><p class="c0"><span class="c1">7. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c0"><span class="c1">8. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.</span></p><p class="c0"><span class="c1">9. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c0"><span class="c1">10. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c0"><span class="c1">11. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><img height="394.0" src="images/image2.png" width="526.0"></p><p class="c0"><span class="c1 c7">Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)</span></p><p class="c0"><span class="c1 c7">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c0"><span class="c1 c7">&nbsp;</span></p><p class="c0"><img height="344.0" src="images/image3.png" width="460.0"></p><p class="c0"><span class="c1 c7">Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)</span></p><p class="c0"><span class="c1 c7">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future-</span></p><p class="c0"><span class="c1 c7">Friday-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c2"><span class="c1">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c2"><span class="c1">if so, continue with strategy. </span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/24/2010</span><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="4"><span class="c1">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c2"><span class="c1">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c2"><span class="c1">if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">1 % Agarose Gel</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">I found that the small gel trays need only 25-30 mL of solution (less than 30)</span></li><li class="c2"><span class="c1">I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it. </span></li><li class="c2"><span class="c1">Gel Start: 9:10 am</span></li><li class="c2"><span class="c1">End 10:10 am</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec of Gel Extract from RFP+HybB PCR </span></p><p class="c0"><span class="c1">19.2 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">(inset gel pic from etbr lab)</span></p><p class="c0"><span class="c1 c7">Gel of Excised 1100 bp band of &nbsp;RFP-FR + HybB FR PCR product </span></p><p class="c0"><span class="c1 c7">4 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)</span></p><p class="c0"><span class="c1">3.5 uL H20</span></p><p class="c0"><span class="c1">3uL 10X Promega Buffer E</span></p><p class="c0"><span class="c1">20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL EcoRI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for pSB1A3 (Debika did this today)</span></p><p class="c0"><span class="c1">3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)</span></p><p class="c0"><span class="c1">5uL 10X Promega Buffer E </span><span class="c1 c10">or Multicore</span></p><p class="c0"><span class="c1">10 uL pSB1A3 (9.16.2010, 100 ng/uL)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL EcoRI</span></p><p class="c0"><span class="c1 c5">Total=50 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)</span></p><p class="c0"><span class="c1">Note- We have had the best results when eluting in </span><span class="c1 c5">30 uL of H20</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purification</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec</span></p><p class="c0"><span class="c1">pSB1A3 Digest (purified) = 6.2 ng/uL</span></p><p class="c0"><span class="c1">RFP+HYBB Digest (purified)= 4.4ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation of pSB1A3 to [RFP-FR+ HybB-FR]</span><span class="c1"> (from 9.23.2010)</span></p><p class="c0"><span class="c1 c7">Calculating equivalents:</span></p><p class="c0"><span class="c1">pSB1A3- [ 6.2 ng/uL]/ 2100 bp= &nbsp;3x10^-3 eq/uL</span></p><p class="c0"><span class="c1">hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3 &nbsp;eq/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">&nbsp;</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)</span></li></ol><ol class="c12"><li class="c6" value="1"><span class="c1">E.g. 1 equivalent of vector to 2 equivalent of linear product</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation Protocol:</span></p><p class="c0"><span class="c1"> 3 uL of RFP + HYB (~ 2 to 3 uL)</span></p><p class="c0"><span class="c1"> 2 uL of &nbsp;Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)</span></p><p class="c0"><span class="c1">1 uL 10x Ligase Buffer</span></p><p class="c0"><span class="c1">3.5 uL H20</span></p><p class="c0"><span class="c1">0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">Total=10 uL</span></p><p class="c0"><span class="c1">Leave RT for 1 hour</span></p><p class="c0"><span class="c1">Start: 5:23 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Transformation</span></p><p class="c0"><span class="c1 c4">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c0"><span class="c1">10 &#1405;L Nova Blue cells + 5 &#1405;L of Ligation Reaction</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for Heat Shock Transformation </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Notes for Future-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Aliquots of BL21 and Novablue are in our -70c freezer</span></li><li class="c2"><span class="c1">Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/25/2010</span></p><p class="c0"><span class="c1 c4">Goals-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Begin digesting the building blocks necessary to create the rest of the constructs</span></li><li class="c2"><span class="c1">Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight</span></li></ol><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec results </span><span class="c1">(From tubes dated 9/17/2010)</span></p><p class="c0"><span class="c1">AOX1a-F,R = 99.3 ng/uL</span></p><p class="c0"><span class="c1">AOX1a-F,R2 = 50.0 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R = 48.5 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R2 = 42.1 ng/uL</span></p><p class="c0"><span class="c1">OmpA-F,R = 27.3 ng/uL</span></p><p class="c0"><span class="c1">RFP-F2,R = 10.8 ng/uL</span></p><p class="c0"><span class="c1">Hyb-F,R = 26.2 ng/uL</span></p><p class="c0"><span class="c1">RFP-F,R = 8.8 ng/uL</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for RFP-F2,R</span></p><p class="c0"><span class="c1">0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)</span></p><p class="c0"><span class="c1">5 uL 10X Promega Buffer D</span></p><p class="c0"><span class="c1">40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL NdeI</span></p><p class="c0"><span class="c1 c5">Total=51.5 ul total </span></p><p class="c0"><span class="c1">Start :4pm</span></p><p class="c0"><span class="c1">End: 7 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for OmpA F,R</span></p><p class="c0"><span class="c1">1.5 uL H20 </span></p><p class="c0"><span class="c1">5 uL 10X Promega Multi-Core Buffer</span></p><p class="c0"><span class="c1">37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NotI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=50 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 2:55 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1a F,R</span></p><p class="c0"><span class="c1">12.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Buffer B</span></p><p class="c0"><span class="c1">10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 3:05 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1a F,R2</span></p><p class="c0"><span class="c1">2.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Multi Core Buffer</span></p><p class="c0"><span class="c1">20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NdeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Start 4 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1b F,R</span></p><p class="c0"><span class="c1">2.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Buffer B</span></p><p class="c0"><span class="c1">20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 3:10 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1b F,R2</span></p><p class="c0"><span class="c1">0 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Multi Core Buffer </span></p><p class="c0"><span class="c1">22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NdeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Start 4 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)</span></p><p class="c0"><span class="c1">End time should be 6,7 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Preparing liquid culture of pSB1A3+hybB+RFP</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010</span></li><li class="c2"><span class="c1">O/N at 37c in our incubator-shaker</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">PCR purify the digests</span></li><li class="c2"><span class="c1">start first round of ligations</span></li><li class="c2"><span class="c1">PCR the ligation products</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">&nbsp;</span></p>
+
<style type="text/css">ol{margin:0;padding:0}p{margin:0}.c6{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c2{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c0{line-height:1.15;text-indent:0pt;direction:ltr}.c8{line-height:1.15;text-indent:36.0pt;direction:ltr}.c1{color:#ffffff;font-size:10pt;font-family:Arial}.c5{font-weight:bold}.c11{background-color:#ffffff}.c4{text-decoration:underline}.c9{list-style-type:decimal}.c10{text-decoration:line-through}.c3{list-style-type:disc}.c13{list-style-type:lower-latin}.c12{list-style-type:circle}.c7{font-style:italic}</style></head><body class="c11"><p class="c0"><span class="c1">Plan for this week:</span></p><p class="c0"><span class="c1">Weekend: PCR purify hybB, run gel for pSB1A3</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Mon: Redo PCR RFP; digest RFP (O/N)</span></li><li class="c2"><span class="c1">Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)</span></li><li class="c2"><span class="c1">Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N</span></li><li class="c2"><span class="c1">Thurs: Tranformation of cells by hyb+RFP+psb1a3</span></li><li class="c2"><span class="c1">Fri: check plates</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/20/2010</span></p><p class="c0"><span class="c1 c4">Goals</span><span class="c1">: </span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Redo PCR of mRFP F,R; mRFP F2,R</span></li><li class="c2"><span class="c1">Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes) </span></li><li class="c2"><span class="c1">RUN GEL - 2 uL each - to check size - this step completed for all building blocks</span></li><li class="c2"><span class="c1">PCR PURIFY leftovers - this step completed for all building blocks</span></li><li class="c2"><span class="c1">NANOSPEC - record concentrations - this step completed for all building blocks</span></li><li class="c2"><span class="c1">Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock</span></li></ol><p class="c0"><span class="c1 c4">Notes:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1 c5">Ryan gave us a fresh stock of mRFP plasmid</span><span class="c1">. </span><span class="c1 c4">We only get this one</span><span class="c1">! It is precious like gold or diamonds!</span></li><li class="c2"><span class="c1">Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest. </span></li></ol><p class="c0"><span class="c1 c4">mRFP</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">in the plasmid PET15B</span></li><li class="c2"><span class="c1">Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c5">9am </span></p><p class="c0"><span class="c1 c4">PCR Protocol </span><span class="c1"> for mRFP F,R; mRFP F2, R</span></p><p class="c0"><span class="c1">26.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c5">PHUSION 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1 c5">2 uL</span><span class="c1"> template DNA</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">PHUSION</span></p><p class="c0"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ran PCR results (pre purification) on gel)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">12pm</span></p><p class="c0"><span class="c1 c4">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c0"><span class="c1">10 &#1405;L Nova Blue cells + 5 &#1405;L of MRFP plasmid (from stock that Ryan gave us this morning).</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for Heat Shock Transformation </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results</span></p><ol class="c9"><li class="c2" value="1"><span class="c1">Gel picture of mRFP FR, F2R (Insert gel pic!)</span></li></ol><ol class="c13"><li class="c6" value="1"><span class="c1">The bands of both PCRs (pre digest) were between approximately around &nbsp;700-800 </span></li></ol><p class="c0"><img height="288.0" src="https://static.igem.org/mediawiki/2010/5/5d/9-19a.png" width="385.0"></p><p class="c0"><span class="c1 c7">Gel of mRFP FR and mRFP F2R</span></p><p class="c0"><span class="c1 c7">100bp ladder|mRFP FR|mRFP F2R|Control</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Purificatio</span><span class="c1">n-mRFP</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purification </span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec Results</span></p><p class="c0"><span class="c1">mRFP, F: 137.2ng/uL</span></p><p class="c0"><span class="c1">mRFP, F2: 132.3 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)</span></li><li class="c2"><span class="c1">Check if transformation of Nova Blue with mRFP worked!</span></li></ol><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/21/2010</span></p><p class="c0"><span class="c1">Results from 9/20/2010</span></p><p class="c0"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transformation of mRFP in NB cells successful (both plates). </span></p><p class="c0"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c0"><span class="c1 c4">Starter Cultures</span></p><p class="c0"><span class="c1">Make starter cultures from both mRFP:NB plates, allow to grow overnight. </span></p><p class="c8"><span class="c1">3mL LB + 3uL 1000X CARB</span></p><p class="c8"><span class="c1">put in 37C incubator with shaking overnight</span></p><p class="c8"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For 9/22/2010:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening</span></li><li class="c2"><span class="c1">Create cryostocks from starter cultures grown on 9/21/2010.</span></li><li class="c2"><span class="c1">Couldn&rsquo;t start RE digest of RFP today- could not find RE&rsquo;s. Will ask Megan Wednesday morning.</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/22/2010- (Mitesh, Scott, Gita)</span></p><p class="c0"><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Digest of RFP F,R</span></li><li class="c2"><span class="c1">PCR purify the digest</span></li><li class="c2"><span class="c1">Ligate hyBB FR, to RFP F,R</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes: made a stock of 1 ug/ul BSA for easier RE digests</span></p><p class="c0"><span class="c1">I am following the suggested RE protocol listed on the Promega literature that came with the RE&rsquo;s</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for RFP-F,R</span><span class="c1">r</span></p><p class="c0"><span class="c1">4.5 uL H20</span></p><p class="c0"><span class="c1">2uL 10X Promega Buffer D</span></p><p class="c0"><span class="c1">10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)</span></p><p class="c0"><span class="c1">2 uL BSA (0ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI (edited changed from notI to speI )</span></p><p class="c0"><span class="c1">0.75 uL NotI</span></p><p class="c0"><span class="c1 c5">Total=20 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">Start time is 10:35 am</span></p><p class="c0"><span class="c1">End time should be 1:35 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Purification of RFP digest</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purfication</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec</span></p><p class="c0"><span class="c1">RFP Digest (purified)- 41 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation of RFP-FR to HybB-FR</span></p><p class="c0"><span class="c1">using hybB from 9/18/2010 and RFP from 9/21/2010</span></p><p class="c0"><span class="c1 c7">Calculating equivalents:</span></p><p class="c0"><span class="c1">RFP- [41 ng/uL]/678 bp= 0.06 eq/uL</span></p><p class="c0"><span class="c1">hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL</span></p><p class="c0"><span class="c1">for linear ligations, use a 1:1 ratio of products</span></p><p class="c0"><span class="c1 c4">Ligation:</span></p><p class="c0"><span class="c1">2 uL of RFP FR (SpeI, NotI digested; purified)</span></p><p class="c0"><span class="c1">2 uL of HybB FR (EcoRI, NotI digested; purified)</span></p><p class="c0"><span class="c1">1 uL 10x Ligase Buffer</span></p><p class="c0"><span class="c1">4.5 uL H20</span></p><p class="c0"><span class="c1">0.5 uL T4 Ligase</span></p><p class="c0"><span class="c1 c5">Total=10 uL</span></p><p class="c0"><span class="c1">Leave RT for 1 hour</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Start time: 3:20 pm</span></p><p class="c0"><span class="c1">End time: 4:20 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR of hybB+Mrfp construct </span></p><p class="c0"><span class="c1">3 uL of ligation reaction</span></p><p class="c0"><span class="c1">25.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c5">PHUSION 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">PHUSION</span></p><p class="c0"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c0"><span class="c1">Start: 5pm</span></p><p class="c0"><span class="c1">End: Thursday</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Take out pcr of mrfp+hybB</span></li><li class="c2"><span class="c1">Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)</span></li><li class="c2"><span class="c1">Digest the construct, along with the vector psb1a3 at the same time (3 hrs)</span></li><li class="c2"><span class="c1">Ligate the construct to psb1a3 (1 hr)</span></li><li class="c2"><span class="c1">Transform into e coli (1.5 hrs)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9.23.2010 (Scott, Rob, Gita)</span></p><p class="c0"><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Run PCR of RFP+hyBb on gel</span></li><li class="c2"><span class="c1">Digest the construct, along with vecotr psb1a3</span></li><li class="c2"><span class="c1">ligate the construct to psb1a3</span></li><li class="c2"><span class="c1">transform into e. coli</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes:</span></p><p class="c0"><span class="c1">Predicted size of hyb+MRFP~1000bp</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Running PCR product on gel</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">The pcr was left overnight at 12 c, and I am running 4 uL on a gel</span></li><li class="c2"><span class="c1">4 &nbsp;uL sample + 1 uL running dye</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Notes:</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.</span></li><li class="c2"><span class="c1">PCR products store in yellow box</span></li><li class="c2"><span class="c1">Building blocks in blue rack</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR</span></p><p class="c0"><span class="c1">1. &nbsp;Heat 30 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">2. Add 35 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">3. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results</span></p><p class="c0"><img height="313.0" src="https://static.igem.org/mediawiki/2010/0/0f/9-19b.png" width="419.0"></p><p class="c0"><span class="c1 c7">Gel from 9.23.2010.</span></p><p class="c0"><span class="c1 c7">100 bp ladder| RFP-FR +HybB PCR|</span></p><p class="c0"><span class="c1 c7">Predicted Size- 1070 bp</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR (Hammer Lab Apparatus)</span></p><p class="c0"><span class="c1">1. &nbsp;Heat 90 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">2. Add 90 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">3. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">(I did 100 mL, but found the actual volume to be around 80 to 90 mL)</span></p><p class="c0"><span class="c1">Start: 4:10 pm</span></p><p class="c0"><span class="c1">End: 5:10 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Gel Extraction Protocol</span></p><p class="c0"><span class="c1">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c0"><span class="c1">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c0"><span class="c1">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c0"><span class="c1">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c0"><span class="c1">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c0"><span class="c1">6. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.</span></p><p class="c0"><span class="c1">7. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c0"><span class="c1">8. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.</span></p><p class="c0"><span class="c1">9. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c0"><span class="c1">10. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c0"><span class="c1">11. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><img height="394.0" src="https://static.igem.org/mediawiki/2010/e/e3/9-19c.png" width="526.0"></p><p class="c0"><span class="c1 c7">Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)</span></p><p class="c0"><span class="c1 c7">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c0"><span class="c1 c7">&nbsp;</span></p><p class="c0"><img height="344.0" src="images/image3.png" width="460.0"></p><p class="c0"><span class="c1 c7">Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)</span></p><p class="c0"><span class="c1 c7">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future-</span></p><p class="c0"><span class="c1 c7">Friday-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c2"><span class="c1">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c2"><span class="c1">if so, continue with strategy. </span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/24/2010</span><span class="c1 c4">Goals</span></p><ol class="c3"><li class="c2" value="4"><span class="c1">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c2"><span class="c1">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c2"><span class="c1">if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">1 % Agarose Gel</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">I found that the small gel trays need only 25-30 mL of solution (less than 30)</span></li><li class="c2"><span class="c1">I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it. </span></li><li class="c2"><span class="c1">Gel Start: 9:10 am</span></li><li class="c2"><span class="c1">End 10:10 am</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec of Gel Extract from RFP+HybB PCR </span></p><p class="c0"><span class="c1">19.2 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">(inset gel pic from etbr lab)</span></p><p class="c0"><span class="c1 c7">Gel of Excised 1100 bp band of &nbsp;RFP-FR + HybB FR PCR product </span></p><p class="c0"><span class="c1 c7">4 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c0"><span class="c1 c7">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)</span></p><p class="c0"><span class="c1">3.5 uL H20</span></p><p class="c0"><span class="c1">3uL 10X Promega Buffer E</span></p><p class="c0"><span class="c1">20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL EcoRI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for pSB1A3 (Debika did this today)</span></p><p class="c0"><span class="c1">3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)</span></p><p class="c0"><span class="c1">5uL 10X Promega Buffer E </span><span class="c1 c10">or Multicore</span></p><p class="c0"><span class="c1">10 uL pSB1A3 (9.16.2010, 100 ng/uL)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL EcoRI</span></p><p class="c0"><span class="c1 c5">Total=50 ul total </span></p><p class="c0"><span class="c1">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)</span></p><p class="c0"><span class="c1">Note- We have had the best results when eluting in </span><span class="c1 c5">30 uL of H20</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for PCR Purification</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec</span></p><p class="c0"><span class="c1">pSB1A3 Digest (purified) = 6.2 ng/uL</span></p><p class="c0"><span class="c1">RFP+HYBB Digest (purified)= 4.4ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation of pSB1A3 to [RFP-FR+ HybB-FR]</span><span class="c1"> (from 9.23.2010)</span></p><p class="c0"><span class="c1 c7">Calculating equivalents:</span></p><p class="c0"><span class="c1">pSB1A3- [ 6.2 ng/uL]/ 2100 bp= &nbsp;3x10^-3 eq/uL</span></p><p class="c0"><span class="c1">hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3 &nbsp;eq/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">&nbsp;</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)</span></li></ol><ol class="c12"><li class="c6" value="1"><span class="c1">E.g. 1 equivalent of vector to 2 equivalent of linear product</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Ligation Protocol:</span></p><p class="c0"><span class="c1"> 3 uL of RFP + HYB (~ 2 to 3 uL)</span></p><p class="c0"><span class="c1"> 2 uL of &nbsp;Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)</span></p><p class="c0"><span class="c1">1 uL 10x Ligase Buffer</span></p><p class="c0"><span class="c1">3.5 uL H20</span></p><p class="c0"><span class="c1">0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">Total=10 uL</span></p><p class="c0"><span class="c1">Leave RT for 1 hour</span></p><p class="c0"><span class="c1">Start: 5:23 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Transformation</span></p><p class="c0"><span class="c1 c4">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c0"><span class="c1">10 &#1405;L Nova Blue cells + 5 &#1405;L of Ligation Reaction</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5 c7">See Protocols page for Heat Shock Transformation </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Notes for Future-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Aliquots of BL21 and Novablue are in our -70c freezer</span></li><li class="c2"><span class="c1">Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c5">9/25/2010</span></p><p class="c0"><span class="c1 c4">Goals-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">Begin digesting the building blocks necessary to create the rest of the constructs</span></li><li class="c2"><span class="c1">Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight</span></li></ol><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec results </span><span class="c1">(From tubes dated 9/17/2010)</span></p><p class="c0"><span class="c1">AOX1a-F,R = 99.3 ng/uL</span></p><p class="c0"><span class="c1">AOX1a-F,R2 = 50.0 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R = 48.5 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R2 = 42.1 ng/uL</span></p><p class="c0"><span class="c1">OmpA-F,R = 27.3 ng/uL</span></p><p class="c0"><span class="c1">RFP-F2,R = 10.8 ng/uL</span></p><p class="c0"><span class="c1">Hyb-F,R = 26.2 ng/uL</span></p><p class="c0"><span class="c1">RFP-F,R = 8.8 ng/uL</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for RFP-F2,R</span></p><p class="c0"><span class="c1">0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)</span></p><p class="c0"><span class="c1">5 uL 10X Promega Buffer D</span></p><p class="c0"><span class="c1">40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL NdeI</span></p><p class="c0"><span class="c1 c5">Total=51.5 ul total </span></p><p class="c0"><span class="c1">Start :4pm</span></p><p class="c0"><span class="c1">End: 7 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for OmpA F,R</span></p><p class="c0"><span class="c1">1.5 uL H20 </span></p><p class="c0"><span class="c1">5 uL 10X Promega Multi-Core Buffer</span></p><p class="c0"><span class="c1">37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)</span></p><p class="c0"><span class="c1">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NotI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=50 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 2:55 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1a F,R</span></p><p class="c0"><span class="c1">12.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Buffer B</span></p><p class="c0"><span class="c1">10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 3:05 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1a F,R2</span></p><p class="c0"><span class="c1">2.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Multi Core Buffer</span></p><p class="c0"><span class="c1">20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NdeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Start 4 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1b F,R</span></p><p class="c0"><span class="c1">2.5 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Buffer B</span></p><p class="c0"><span class="c1">20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL SpeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c5">Went in at 3:10 pm</span></p><p class="c0"><span class="c1 c5">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Double Digest Recipe for AOX1b F,R2</span></p><p class="c0"><span class="c1">0 uL H20 </span></p><p class="c0"><span class="c1">3 uL 10X Promega Multi Core Buffer </span></p><p class="c0"><span class="c1">22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)</span></p><p class="c0"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c0"><span class="c1">0.75 uL NdeI </span></p><p class="c0"><span class="c1">0.75 uL XmaI</span></p><p class="c0"><span class="c1 c5">Total=30 ul total </span></p><p class="c0"><span class="c1">Start 4 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c0"><span class="c1">Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)</span></p><p class="c0"><span class="c1">End time should be 6,7 pm</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Preparing liquid culture of pSB1A3+hybB+RFP</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010</span></li><li class="c2"><span class="c1">O/N at 37c in our incubator-shaker</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future-</span></p><ol class="c3"><li class="c2" value="1"><span class="c1">PCR purify the digests</span></li><li class="c2"><span class="c1">start first round of ligations</span></li><li class="c2"><span class="c1">PCR the ligation products</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">&nbsp;</span></p>

Revision as of 19:18, 24 October 2010

Georgia Institute of Technology iGEM Team 2010 Homepage

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Plan for this week:

Weekend: PCR purify hybB, run gel for pSB1A3

  1. Mon: Redo PCR RFP; digest RFP (O/N)
  2. Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)
  3. Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N
  4. Thurs: Tranformation of cells by hyb+RFP+psb1a3
  5. Fri: check plates

 

9/20/2010

Goals:

  1. Redo PCR of mRFP F,R; mRFP F2,R
  2. Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes)
  3. RUN GEL - 2 uL each - to check size - this step completed for all building blocks
  4. PCR PURIFY leftovers - this step completed for all building blocks
  5. NANOSPEC - record concentrations - this step completed for all building blocks
  6. Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock

Notes:

  1. Ryan gave us a fresh stock of mRFP plasmid. We only get this one! It is precious like gold or diamonds!
  2. Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest.

mRFP

  1. in the plasmid PET15B
  2. Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)

 

Protocols

 

9am

PCR Protocol for mRFP F,R; mRFP F2, R

26.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

 

Ran PCR results (pre purification) on gel)

 

12pm

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of MRFP plasmid (from stock that Ryan gave us this morning).

 

See Protocols page for Heat Shock Transformation

 

Results

  1. Gel picture of mRFP FR, F2R (Insert gel pic!)
  1. The bands of both PCRs (pre digest) were between approximately around  700-800

Gel of mRFP FR and mRFP F2R

100bp ladder|mRFP FR|mRFP F2R|Control

 

PCR Purification-mRFP

See Protocols page for PCR Purification

 

Nanospec Results

mRFP, F: 137.2ng/uL

mRFP, F2: 132.3 ng/uL

 

For Future

  1. Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)
  2. Check if transformation of Nova Blue with mRFP worked!

 

9/21/2010

Results from 9/20/2010

        Transformation of mRFP in NB cells successful (both plates).

        

Starter Cultures

Make starter cultures from both mRFP:NB plates, allow to grow overnight.

3mL LB + 3uL 1000X CARB

put in 37C incubator with shaking overnight

 

Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).

 

For 9/22/2010:

  1. Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening
  2. Create cryostocks from starter cultures grown on 9/21/2010.
  3. Couldn’t start RE digest of RFP today- could not find RE’s. Will ask Megan Wednesday morning.

 

9/22/2010- (Mitesh, Scott, Gita)

Goals

  1. Digest of RFP F,R
  2. PCR purify the digest
  3. Ligate hyBB FR, to RFP F,R

 

Notes: made a stock of 1 ug/ul BSA for easier RE digests

I am following the suggested RE protocol listed on the Promega literature that came with the RE’s

 

RE Digest Recipe for RFP-F,Rr

4.5 uL H20

2uL 10X Promega Buffer D

10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)

2 uL BSA (0ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI (edited changed from notI to speI )

0.75 uL NotI

Total=20 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 10:35 am

End time should be 1:35 pm

 

Purification of RFP digest

See Protocols page for PCR Purfication

 

Nanospec

RFP Digest (purified)- 41 ng/uL

 

Ligation of RFP-FR to HybB-FR

using hybB from 9/18/2010 and RFP from 9/21/2010

Calculating equivalents:

RFP- [41 ng/uL]/678 bp= 0.06 eq/uL

hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

2 uL of RFP FR (SpeI, NotI digested; purified)

2 uL of HybB FR (EcoRI, NotI digested; purified)

1 uL 10x Ligase Buffer

4.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

 

Start time: 3:20 pm

End time: 4:20 pm

 

PCR of hybB+Mrfp construct

3 uL of ligation reaction

25.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 5pm

End: Thursday

 

For Future

  1. Take out pcr of mrfp+hybB
  2. Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)
  3. Digest the construct, along with the vector psb1a3 at the same time (3 hrs)
  4. Ligate the construct to psb1a3 (1 hr)
  5. Transform into e coli (1.5 hrs)

 

9.23.2010 (Scott, Rob, Gita)

Goals

  1. Run PCR of RFP+hyBb on gel
  2. Digest the construct, along with vecotr psb1a3
  3. ligate the construct to psb1a3
  4. transform into e. coli

 

Notes:

Predicted size of hyb+MRFP~1000bp

 

Protocols

 

Running PCR product on gel

  1. The pcr was left overnight at 12 c, and I am running 4 uL on a gel
  2. 4  uL sample + 1 uL running dye

 

Notes:

  1. I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.
  2. PCR products store in yellow box
  3. Building blocks in blue rack

 

Making gel for PCR

1.  Heat 30 mL of 1% agarose solution  in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

2. Add 35 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

 

Results

Gel from 9.23.2010.

100 bp ladder| RFP-FR +HybB PCR|

Predicted Size- 1070 bp

 

Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier.

 

Making gel for PCR (Hammer Lab Apparatus)

1.  Heat 90 mL of 1% agarose solution  in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

2. Add 90 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

(I did 100 mL, but found the actual volume to be around 80 to 90 mL)

Start: 4:10 pm

End: 5:10 pm

 

Gel Extraction Protocol

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.

7. Discarded flow-through and placed QIAquick column back in the same collection tube.

8. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.

9. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

10. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

11. To elute DNA, added 30  μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min.

 

Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

 

Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

 

For Future-

Friday-

  1. nanospec the gel extracted hyb+RFP construct (in blue box)
  2. Run 2 uL on gel, see if 1100 bp is confirmed
  3. if so, continue with strategy.

 

9/24/2010Goals

  1. nanospec the gel extracted hyb+RFP construct (in blue box)
  2. Run 2 uL on gel, see if 1100 bp is confirmed
  3. if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)

 

Protocols

 

1 % Agarose Gel

  1. I found that the small gel trays need only 25-30 mL of solution (less than 30)
  2. I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it.
  3. Gel Start: 9:10 am
  4. End 10:10 am

 

Nanospec of Gel Extract from RFP+HybB PCR

19.2 ng/uL

 

(inset gel pic from etbr lab)

Gel of Excised 1100 bp band of  RFP-FR + HybB FR PCR product

4 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

 

RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)

3.5 uL H20

3uL 10X Promega Buffer E

20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=30 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

 

RE Digest Recipe for pSB1A3 (Debika did this today)

3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)

5uL 10X Promega Buffer E or Multicore

10 uL pSB1A3 (9.16.2010, 100 ng/uL)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=50 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

 

PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)

Note- We have had the best results when eluting in 30 uL of H20

See Protocols page for PCR Purification

 

Nanospec

pSB1A3 Digest (purified) = 6.2 ng/uL

RFP+HYBB Digest (purified)= 4.4ng/uL

 

Ligation of pSB1A3 to [RFP-FR+ HybB-FR] (from 9.23.2010)

Calculating equivalents:

pSB1A3- [ 6.2 ng/uL]/ 2100 bp=  3x10^-3 eq/uL

hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3  eq/uL

 

 

  1. For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)
  1. E.g. 1 equivalent of vector to 2 equivalent of linear product

 

Ligation Protocol:

3 uL of RFP + HYB (~ 2 to 3 uL)

2 uL of  Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)

1 uL 10x Ligase Buffer

3.5 uL H20

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

 

Total=10 uL

Leave RT for 1 hour

Start: 5:23 pm

 

Transformation

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of Ligation Reaction

 

See Protocols page for Heat Shock Transformation

 

Notes for Future-

  1. Aliquots of BL21 and Novablue are in our -70c freezer
  2. Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want

 

9/25/2010

Goals-

  1. Begin digesting the building blocks necessary to create the rest of the constructs
  2. Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight

 

Nanospec results (From tubes dated 9/17/2010)

AOX1a-F,R = 99.3 ng/uL

AOX1a-F,R2 = 50.0 ng/uL

AOX1b-F,R = 48.5 ng/uL

AOX1b-F,R2 = 42.1 ng/uL

OmpA-F,R = 27.3 ng/uL

RFP-F2,R = 10.8 ng/uL

Hyb-F,R = 26.2 ng/uL

RFP-F,R = 8.8 ng/uL

 

RE Double Digest Recipe for RFP-F2,R

0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)

5 uL 10X Promega Buffer D

40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL NdeI

Total=51.5 ul total

Start :4pm

End: 7 pm

 

RE Double Digest Recipe for OmpA F,R

1.5 uL H20

5 uL 10X Promega Multi-Core Buffer

37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NotI

0.75 uL XmaI

Total=50 ul total

 

Went in at 2:55 pm

 

RE Double Digest Recipe for AOX1a F,R

12.5 uL H20

3 uL 10X Promega Buffer B

10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

 

Went in at 3:05 pm

 

RE Double Digest Recipe for AOX1a F,R2

2.5 uL H20

3 uL 10X Promega Multi Core Buffer

20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

 

RE Double Digest Recipe for AOX1b F,R

2.5 uL H20

3 uL 10X Promega Buffer B

20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

 

Went in at 3:10 pm

 

RE Double Digest Recipe for AOX1b F,R2

0 uL H20

3 uL 10X Promega Multi Core Buffer

22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

 

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)

End time should be 6,7 pm

 

Preparing liquid culture of pSB1A3+hybB+RFP

  1. 3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010
  2. O/N at 37c in our incubator-shaker

 

For Future-

  1. PCR purify the digests
  2. start first round of ligations
  3. PCR the ligation products