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JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.
Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:
From the 2010 Parts Distribution:
(In Lab: JV)
Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.
Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).
Restriction Reaction
Ingredient | Volume(µL) |
MilliQ H20 Water | 15.75 |
Orange Buffer (10x) | 2 |
pDNA (rbs-xylE) | 2 |
EcoRI | 0.25 |
Unrestricted Control
Ingredient | Volume(µL) |
MilliQ H20 Water | 16 |
Orange Buffer (10x) | 2 |
pDNA (rbs-xylE) | 2 |
DNA was restricted for 80 minutes at 37oC.
Analyzed results on a 1% agarose gel. Load order as follows:
Lane | Sample | Volume Sample (µL) | Volume Loading Dye (µL) |
1 | Restricted RBS-xylE | 10 | 2 |
1 | Unestricted RBS-xylE† | 1 | 2 |
1 | 1kb Ladder†† | 2 | 2 |
† Added 9µL MilliQ H2O
†† Added 8µL MilliQ H2O
Ran gel at 100V from 2 hours.
Results:
Conclusions: Plasmid DNA prep and restriction was successful.
Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:
Component | Volume (µL) |
MilliQ H2O | 15.5 |
Buffer | 2 |
pDNA | 2 |
Enzyme | 0.25 + 0.25 |
Set up control reaction as follows:
Incubated reactions for 65 minutes at 37oC
Killed enzymes by incubating reactions for 10 minutes at 65oC
Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
Relevant Information:
Common Name | Location | Concentration (ng/µL) | Volume/rxn (µL) |
pLacI Maxiprep | A9 | 990 | ~1 |
pLacI (B1) | A6 | 440 | ~2 |
sRBS-Lum-dT (2) | A1 | 965 | ~1 |
sRBS-Lum-dT (1) | A2 | 1145 | ~1 |
sRBS-Lum-dT Maxiprep | B8 | 4780 | ~.2 |
sRBS-Lum-dT | B7 | 4375 | ~.25 |
sRBS-Lum-dT (1) | G2 | 335 | ~3 |
sRBS-Lum-dT (2) | G3 | 965 | ~2 |
Method:
Restriction
Name | [pDNA] (ng/µL) | Volume pDNA (µL) | Volume Water (µL) | Volume Buffer (µL) | Enzymes | Total Volume |
sRBS-Lum-dT (A1) | 965 | 1 | 43.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
sRBS-Lum-dT (A2) | 1145 | 1 | 43.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pLacI Maxiprep (A1) | 990 | 1 | 43.5 | 5 | 0.25µL EcoRI 0.25µL SpeI | 50 |
sRBS-Lum-dT Maxiprep(B8) | 4780 | 2 (of 1:10 dilution) | 42.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
sRBS-Lum-dT (B7) | 4375 | 2.5 (of 1:10 dilution) | 42 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pLacI (D6) | 440 | 2 | 42.5 | 5 | 0.25µL EcoRI 0.25µL SpeI | 50 |
sRBS-Lum-dT (G2) | 335 | 3 | 41.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
sRBS-Lum-dT (G3) | 540 | 2 | 42.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pSB1T3 | 25 | 12.5 | 7 | 5 | 0.25µL EcoRI 0.25µL PstI | 50 |
Incubate for 30 minutes at 37oC (Start- 12:10pm; End- 12:40pm)
Heat kill enzymes at 80oC for 20 minutes
Ligation:
In a 10µL final volume, add:
Incubate for 30 minutes at room temperature to ligate
Incubate for 20 minutes at 80oC to heat kill
Carried out protocol described in June 2/2010 - Evening
Analyzed results on 1% agarose gel.Load order as follows:
Lane | Gel 1 Sample | Gel 1 Load | Gel 2 Sample | Gel 2 Load |
1 | 1kb Ladder | 2µL dye, 2µL ladder 8µL MilliQ H2O | 1kb Ladder | 2µL dye, 2µL ladder 8µL MilliQ H2O |
2 | Restricted sRBS-Lum-dT (A1) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A1) | 10µL DNA 2µL Dye |
3 | Unrestricted sRBS-Lum-dT (A1) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A1) | 10µL DNA 2µL Dye |
4 | Restricted sRBS-Lum-dT (A2) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A2) | 10µL DNA 2µL Dye |
5 | Unrestricted sRBS-Lum-dT (A2) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A2) | 10µL DNA 2µL Dye |
6 | Restricted sRBS-Lum-dT (B8) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B7) | 10µL DNA 2µL Dye |
7 | Unrestricted sRBS-Lum-dT (B8) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B7) | 10µL DNA 2µL Dye |
8 | Restricted sRBS-Lum-dT (B7) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G2) | 10µL DNA 2µL Dye |
9 | Unrestricted sRBS-Lum-dT (B7) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G2) | 10µL DNA 2µL Dye |
10 | Restricted sRBS-Lum-dT (G2) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G3) | 10µL DNA 2µL Dye |
11 | Unrestricted sRBS-Lum-dT (G2) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G3) | 10µL DNA 2µL Dye |
12 | Restricted sRBS-Lum-dT (G3) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B8) | 10µL DNA 2µL Dye |
13 | Unrestricted sRBS-Lum-dT (G3) | 10µL DNA 2µL Dye | pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B8) | 10µL DNA 2µL Dye |
14 | Restricted pLacI (A9) | 10µL DNA 2µL Dye | Restricted rbs-xylE | 10µL DNA 2µL Dye |
15 | Unrestricted pLacI (A9) | 10µL DNA 2µL Dye | Unrestricted rbs-xylE | 10µL DNA 2µL Dye |
16 | Restricted pLacI B1 (D6) | 10µL DNA 2µL Dye | Restricted pSB1T3 | 10µL DNA 2µL Dye |
17 | Unrestricted pLacI B1 (D6) | 10µL DNA 2µL Dye | Unrestricted pSB1T3 | 10µL DNA 2µL Dye |
18 | Unrestricted dT | 10µL DNA 2µL Dye | pSB1T3 Ligation of: rbs-xylE+dT | 10µL DNA 2µL Dye |
19 | Restricted dT | 10µL DNA 2µL Dye |
Ran gel at 100V for 90 minutes.
Results:
Conclusions:
Objective: Repeat restriction of pSB1T3 and ligate with pLacI and sRBS-Lum-dT. Previous ligations all used up on gel.
Method:
Ligation:
In a 10µL final volume, add:
Incubate for 30 minutes at room temperature to ligate
Incubate for 20 minutes at 80oC to heat kill
Following ligation, transformed using transformation protocol.
Plates incubated in 37oC incubator for 44 hours.
Results: Only plate pLacI (D6) + sRBS-Lum-dT (G2) + pSB1T3 grew; had 2 colonies. Control plate did not grow, acidentally plated on tetracycline plate instead of ampicillin (pUC19).
Follow-up: Inoculated 5mL LB media (tetracycline positive) with cells from the transformation plates and incubated at 37oC overnight. (June 5/2010).
Objective: Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.
Method:
Restriction
Name | Volume pDNA (µL) | Volume Water (µL) | Volume Buffer (µL) | Enzymes | Total Volume |
pSB-NEYFP (B4) | .8 | 43.7 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pSB-CEYFP (B5) | .9 | 43.6 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pBad-TetR) | .3 | 44.2 | 5 | 0.25µL EcoRI 0.25µL SpeI | 50 |
NEYFP (E1) | 4.3 | 40.7 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
NEYFP (E2) | 0.3 | 42.2 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
Fusion CEYFP (E3) | 3.9 | 40.6 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
Fusion CEYFP (E4) | 2.0 | 42.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
Fusion CEYFP (E5) | 3.0 | 41.5 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
CEYFP (E6) | 0.6 | 43.9 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
CEYFP (E7) | 0.5 | 44.0 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pBad-TetR (F4) | 2.5 | 42 | 5 | 0.25µL EcoRI 0.25µL SpeI | 50 |
pBad-TetR (F5) | 1.7 | 42.8 | 5 | 0.25µL EcoRI 0.25µL SpeI | 50 |
pSB-CEYFP (G4) | 2.9 | 41.6 | 5 | 0.25µL XbaI 0.25µL PstI | 50 |
pSB1C3 | 15.5 | 46 | 0.25µL EcoRI 0.25µL PstI | 62 |
Incubated at 37oC for 75 minutes.
Continue Ligation on Saturday (See below).
(In the lab:AS)
Objective: Ligate restriction products from June 3/2010.
Relevant information:
Method:
*Restriction digests were not heat killed after reactions. Freezing probably killed the restriction enzymes, but I will hea kill them at 80oC for 20 minutes anyways prior to adding Ligase.
Master Mix | Volume/tube (µL) | Total Volume (µL) |
DNA | 6 | --- |
10x Buffer | 1 | 32 |
T4 DNA Ligase | .25 | 8 |
MilliQ H2O | 2.75 | 88 |
Follow-up: Ligation reactions will be transformed into DH5α cells
(In Lab: JV, HS)
Objective:
Isolate the following plasmid DNA from DH5α:
Method:
Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H2O and RNase A.
Notes:
Objective:
Transformed the following plasmid DNA into DH5α cells:
Method:
Followed Competent Cell Transformation protocol and used chloramphenicol as an antibiotic. We plated all 200µL of DNA onto the plates. The plates were incubated at 37oC from 4:30pm to 10:00am.
Results:
None of the plates showed any growth.
(In Lab: JV, HB, TF, AV)
Objective:
Purification of DNA to increase ligation and transformation efficiency.
Method:
BioBasic Protocol for Purification of PCR products.
DNA (from Working Plasmid Box) Purified:
Objective:
Restrict and run 1% agarose gel of plasmids J5, J6, and pSB1T3 (June 2/10).
Method:
Restrictions
Component | Volume (µL) |
Restriction Enzyme (XbaI) | 0.25 |
Buffer (Tango) | 2 |
Plasmid DNA | 2 |
MilliQ H2O | 15.75 |
Incubated for 1 hour in 37oC incubator.
Heat shocked for 10 min at 65oC on heating block.
1% Agarose Gel
Lane | Sample | Load (µL) |
1 | 1kb ladder | 2 ladder + 2 dye (6X) + 8 MilliQ H2O |
2 | J5-pLacI-SRBS-Lumazine-dT(1) | 10 DNA + 2 Dye (6X) |
3 | J5-pLacI-SRBS-Lumazine-dT(1)restricted | 10 DNA + 2 Dye (6X) |
4 | J6-pLacI-SRBS-Lumazine-dT(2) | 10 DNA + 2 Dye (6X) |
5 | J5-pLacI-SRBS-Lumazine-dT(2)restricted | 10 DNA + 2 Dye (6X) |
6 | pSB1T3 | 10 DNA + 2 Dye (6X) |
7 | pSB1T3 restricted | 10 DNA + 2 Dye (6X) |
8 | Empty | |
9 | Empty | |
10 | Empty |
Ran at 100V for 72 min.
IMAGE TO COME!!!!!!
(In Lab: AS, KG)
Objective:
Ligation of:
Method:
FILL ME IN!!!!!!
(In the lab: JV, AV)
Objective: Follow the overexpression of our pLacI-sRBS-Lum-dT construct.
Method: FILL ME OUT!!!!!!
Results:
Time | OD600 F1 | OD600 E10 |
0 | 0.118 | 0.103 |
30 | 0.133 | 0.111 |
30 | 0.145 | 0.116 |
90 | ||
120 | 0.160 | 0.124 |
150 | 0.120 | 0.093 |
180 | 0.129 | 0.100 |
210 | 0.145 | 0.122 |
240 | 0.158 | 0.145 |
270 | 0.171 | 0.178 |
300 | 0.194 | 0.222 |
330 | 0.223 | 0.280 |
360 | 0.252 | 0.364 |
390 | 0.296 | 0.458 |
420 | 0.338 | 0.557† |
450 | 0.394 | 0.656 |
480 | 0.453 | 0.675 |
510 | 0.530 | 0.688 |
540 | 0.598† | 0.706 |
600 | 0.633 | 0.752 |
660 | 0.653 | |
720 | 0.679 | |
∞ | 1.278 |
† Overexpression induced by adding 1mM IPTG.
Following overexpression, 1mL of cells was removed from the culture, spun down at ~13000xg for 20 seconds, excess media removed and rinsed with water.
Suspended cells in 8M urea, mixed with 6x dye and ran on 18% SDS-PAGE gel for 90 minutes at 200V. Gel stained overnight.
Results: IMAGE TO COME!!!!
Objective: Calculate quantity of DNA in pBad-TetR and fluorescent protein mini-preps by staining an agarose gel.
Method: Restrict plasmid DNA (done by AV,HB,TF on June 7/2010) and run on a 1% TAE agarose gel (JV).
Lane | Gel 1 Sample | Gel 1 Load | Gel 2 Sample | Gel 2 Load |
1 | 1kb Ladder | 2µL dye, 2µL ladder 8µL MilliQ H2O | 1kb Ladder | 2µL dye, 2µL ladder 8µL MilliQ H2O |
2 | Restricted EYFP (B1) | 10µL DNA 2µL Dye | Restricted Fusion CEYFP (E3) | 10µL DNA 2µL Dye |
3 | Unrestricted EYFP (B1) | 10µL DNA 2µL Dye | Unrestricted Fusion CEYFP (E3) | 10µL DNA 2µL Dye |
4 | Restricted pSB-CEYFP (B5) | 10µL DNA 2µL Dye | Restricted Fusion CEYFP (E4) | 10µL DNA 2µL Dye |
5 | Unrestricted pSB-CEYFP (B5) | 10µL DNA 2µL Dye | Unrestricted Fusion CEYFP (E4) | 10µL DNA 2µL Dye |
6 | Restricted ECFP (F2) | 10µL DNA 2µL Dye | Restricted EYFP (E10) | 10µL DNA 2µL Dye |
7 | Unrestricted ECFP (F2) | 10µL DNA 2µL Dye | Unrestricted EYFP (E10) | 10µL DNA 2µL Dye |
8 | Restricted pSB-CEYFP (G4) | 10µL DNA 2µL Dye | Restricted pSB NEYFP (B4) | 10µL DNA 2µL Dye |
9 | Unrestricted pSB-CEYFP (G4) | 10µL DNA 2µL Dye | Unrestricted pSB NEYFP (B4) | 10µL DNA 2µL Dye |
10 | Restricted EYFP (G1) | 10µL DNA 2µL Dye | Restricted ECFP (F3) | 10µL DNA 2µL Dye |
11 | Unrestricted EYFP (G1) | 10µL DNA 2µL Dye | Unrestricted ECFP (F3) | 10µL DNA 2µL Dye |
12 | Restricted NEYFP (E2) | 10µL DNA 2µL Dye | pBad-TetR (F5) | 10µL DNA 2µL Dye |
13 | Unrestricted NEYFP (E2) | 10µL DNA 2µL Dye | Restricted Fusion CEYFP (E5) | 10µL DNA 2µL Dye |
14 | Restricted pBad-TetR (B10) | 10µL DNA 2µL Dye | Unrestricted Fusion CEYFP (E5) | 10µL DNA 2µL Dye |
15 | Unrestricted pBad-TetR (B10) | 10µL DNA 2µL Dye | pBad-TetR (F4) | 10µL DNA 2µL Dye |
16 | Restricted CEYFP (E6) | 10µL DNA 2µL Dye | Restricted pSB1T3 | 10µL DNA 2µL Dye |
17 | Unrestricted CEYFP (E6) | 10µL DNA 2µL Dye | Unrestricted pSB1T3 | 10µL DNA 2µL Dye |
18 | Restricted NEYFP (E1) | 10µL DNA 2µL Dye | 10µL DNA 2µL Dye | |
19 | Unrestricted NEYFP (E1) | 10µL DNA 2µL Dye | ||
20 | Restricted ECFP (F1) | 10µL DNA 2µL Dye | ||
21 | Unrestricted ECFP (F1) | 10µL DNA 2µL Dye | ||
22 | Restricted EYFP (E9) | 10µL DNA 2µL Dye | ||
23 | Unrestricted EYFP (E9) | 10µL DNA 2µL Dye | ||
24 | Restricted CEYFP (E7) | 10µL DNA 2µL Dye | ||
25 | Unrestricted CEYFP (E7) | 10µL DNA 2µL Dye | ||
26 | Restricted EYFP (E8) | 10µL DNA 2µL Dye | ||
27 | Unrestricted EYFP (E8) | 10µL DNA 2µL Dye |
Ran gel at 100V for 90 minutes.
Results:
No bands visible except for pSB1T3 lanes, therefore could not quantify anything that had not already been quantified.
Also, purification of DNA done on June 7/2010 seemed to reduce amount of pDNA in sample.
Objective: Restrict mms6 (D9,D10) and dT (C1) so we can ligate the dT onto the mms6 coding region.
Method:
mms6 Restriction
dT Restriction
Incubated for 1 hour at 37oC
Heat shock on heat block (80oC) for 20 minutes
Ligation
Analyze results on 1% TAE agarose gel
Lane | Sample | Load (µL) |
1 | 1kb ladder | 0.5 ladder; 2 dye; 9.5 MilliQ H2O |
2 | Unrestricted mms6 (D9) | 10 DNA; 2 Dye |
3 | Restricted mms6 (D9) | 10 DNA; 2 Dye |
4 | Unrestricted mms6 (D10) | 10 DNA; 2 Dye |
5 | Restricted mms6 (D10) | 10 DNA; 2 Dye |
6 | Unrestricted dT (C1) | 10 DNA; 2 Dye |
7 | Restricted dT (C1) | 10 DNA; 2 Dye |
8 | Empty | |
9 | Empty | |
10 | Empty |
Results:
Looks like the mms6 DNA was not cut at all. Therefore is doubtful that the ligations will work.
Objective: Transform pLacI-sRBS-Lum-dT constructs assembled via three antibiotic assembly on June 3/2010. Also transform mms6-dT constructs assembled today using old insertion method.
Method: Follow transformation protocol.
Results: No colonies anywhere
(in the lab: TF, JV)
Objective: Transform mms6-dT ligation reactions from June 8/2010.
Method: Followed transformation protocol and transformed the following:
Results: No transformants on plates.
(In the lab: AV, HB, JV)
Objective: Repeat ligation of mms6 (B9,D9,D10) and dT (C1).
Method:
mms6 Restriction
dT Restriction
Incubated for 1 hour at 37oC.
Killed enzymes by heating to 80oC for 20 minutes
Ligation
Incubated at room temperature overnight.
Results:
Analyzed on 1% TAE agarose gel:
Lane | Sample | Load (µL) |
1 | 1kb ladder | 0.5 ladder; 2 dye; 9.5 MilliQ H2O |
2 | Unrestricted mms6 (B9) | 5 DNA; 2 dye; 5 MilliQ H2O |
3 | Restricted mms6 (B9) | 5 DNA; 2 dye; 5 MilliQ H2O |
4 | Unrestricted mms6 (D9) | 5 DNA; 2 dye; 5 MilliQ H2O |
5 | Restricted mms6 (D9) | 5 DNA; 2 dye; 5 MilliQ H2O |
6 | Unrestricted mms6 (D10) | 5 DNA; 2 dye; 5 MilliQ H2O |
7 | Restricted mms6 (D10) | 5 DNA; 2 dye; 5 MilliQ H2O |
8 | Unrestricted dT (C1) | 5 DNA; 2 dye; 5 MilliQ H2O |
9 | Restricted dT (C1) | 5 DNA; 2 dye; 5 MilliQ H2O |
10 | D9 + C1 Ligation (June 8/2010) | 5 DNA; 2 dye; 5 MilliQ H2O |
Gel run for 80 minutes at 100V
Looks like everything was restricted. Ligations may work this time.
Follow-up: Ligation mixtures can be restriction tested and (if cutting works) transformed into DH5α cells.
Objective: Transform pDNA from distribution plates into DH5α cells to generate glycerol stocks for lab.
Method:
Remove DNA from the following locations on the 2010 distributions kits:
Transform into DH5α cells using transformation protocol, with pUC19 as positive control (1ng/µL), and water as negative control.
Results:
(In the lab: TF, DM, HS)
Objective: Restriction Digest of RBS-xylE with EcoRI and SpeI
Method:
Restriction Digestions
Objective: Test Ligations of rbs-xylE with T4-Ligase from iGEM -20oC and from HJ's lab's -20oC.
Method:
(In the lab: JV)
Objective:Continue T4-Ligase check and confirm that it is functional.
Method:Run plasmid DNA;uncut, cut, and ligated on a 1% agarose gel (TAE)
Lane | Sample | Load (µL) |
1 | rbs-xylE | 10 DNA solution + 2 Dye |
2 | rbs-xylE R.D. | 10 DNA solution + 2 Dye2O |
3 | rbs-xylE [iGEM ligation] | 10 DNA solution + 2 Dye |
4 | rbs-xylE [HJ ligation] | 10 DNA solution + 2 Dye |
5 | rbs-xylE [iGEM ligation control] | 10 DNA solution + 2 Dye |
6 | rbs-xylE [HJ ligation control] | 10 DNA solution + 2 Dye |
7 | 1kb ladder | 2 ladder + 2 dye + 8 Milli-Q H2O |
8 | mms6 (B9) R.D. | 10 DNA solution + 2 Dye |
9 | mms6 (B9) | 10 DNA solution + 2 Dye |
10 | mms6 (D10) R.D. | 10 DNA solution + 2 Dye |
11 | mms6 (D10) | 10 DNA solution + 2 Dye |
12 | mms6 (D9) R.D. | 10 DNA solution + 2 Dye |
13 | mms6 (D19) | 10 DNA solution + 2 Dye |
14 | dt (C1) R.D. | 10 DNA solution + 2 Dye |
15 | dt (C1) | 10 DNA solution + 2 Dye |
Result:Ran gel for 89 minutes at 100V. Gel was unreadable. I will redo the gel using and 8 well comb. IMAGE TO COME!!!!
Lane | Sample | Load (µL) |
1 | rbs-xylE R.D. | 5 DNA solution + 1 Dye |
2 | rbs-xylE | 5 DNA solution + 1 Dye |
3 | iGEM ligation | 10 DNA solution + 2 Dye |
4 | iGEM ligation control | 10 DNA solution + 2 Dye |
5 | HJ ligation | 10 DNA solution + 2 Dye |
6 | HJ ligation control | 10 DNA solution + 2 Dye |
7 | 1kb ladder | 2 ladder + 2 dye + 8 Milli-Q H2O |
Ran gel at 100V for 82 minutes.
Result:The gel "broke" while running. Jeff hypothesized the cause to be excessive heat. However, the DNA was still visible.IMAGE TO COME!!!!
Objective:Isolate plasmid DNA from DH5α cells.
Method:The plasmid DNA is:
This will give us 2 additional dt's (3 total) into our working plasmid box to ligate onto the end of our constructs.
Used the boiling lysis miniprep protocol and elute with 50µL of Milli-Q H2O with RNAse A.
These plasmids (labeled 1 & 2 above) are in:
DNA was then purified using the protocol for purification of PCR products (BioBasic). DNA was eluted with 35µL of elution buffer.
(In the lab: AS)
Adam: not convinced that the rbs-xylE was cut properly last night. There doesn't appear to be and band where the insert should be.
Objective: To confirm that EcoRI & SpeI are cutting (also PstI). Use rbs-xylE as test plasmid DNA.
Method: Digest rbs-xylE with the following enzyme combinations:
Reaction mix as follows:
Incubated at 37oC for 1 hour . Analyze on 1% TAE Agarose gel as follows:
Lane | Sample | Load (µL) |
1 | No Enzyme Control (Tango) | 10 DNA solution + 2 Dye |
2 | No Enzyme Control (Red) | 10 DNA solution + 2 Dye |
3 | PstI | 10 DNA solution + 2 Dye |
4 | SpeI (old) | 10 DNA solution + 2 Dye |
5 | SpeI (new) | 10 DNA solution + 2 Dye |
6 | EcoRI | 10 DNA solution + 2 Dye |
7 | EcoRI + SpeI (old) | 10 DNA solution + 2 Dye |
8 | EcoRI + SpeI (new) | 10 DNA solution + 2 Dye |
9 | EcoRI + PstI | 10 DNA solution + 2 Dye |
10 | 1kb ladder | 0.5 ladder + 2 dye + 9.5 Milli-Q H2Oe |
Results: Everything cuts exactly as it should. Continue with ligation and analysis. IMAGE TO COME!!!!
(in lab: JV)
Objective: To miniprep Adam's overnight cultures of xylE and rbs. Completing this will give us a working stock of this plasmid.
Method: Use the boiling lysis miniprep protocol and pufrify using BioBasic protocol for purification of PCR products.
Objective: Transform pLacI-sRBS-lumazine synthase-dt into BL21(DE3).
Method:
Changes to the protocol include:
Results: There wasn't any growth after overnight incubation. Possibility of an antibiotic mix-up.
(in lab: ADS)
Objective: Continue with ligation test. Use restriction products to test T4 DNA ligase.
Method: Have 10µL of DNA remaining for each restriction. Ligate together one of the single cut reactions, and ligate one of the double cut reactions. 4µL of each sample will be tested against each ligase.
i.e.
Reaction Mixture:
Prior to setting up reaction, heat kill restriction enzymes by heating to 80oC for 20 minutes and subsequently cooling on ice for 10 minutes.
Ligations begun at 6:50pm
Will run samples at 30 minutes reaction time and overnight.
Analyze ligations in a 1% TAE Agarose gel
Lane | Sample | Load (µL) |
1 | No Enzyme Control (from last night) | 1 DNA solution + 1 Dye + 4 H2O |
2 | EcoRI + SpeI (old)(from last night) | 1 DNA solution + 1 Dye + 4 H2O |
3 | EcoRI + SpeI (new) vs HJ's Ligase | 5 DNA solution + 1 Dye |
4 | EcoRI + SpeI (new) vs iGEM ligase | 5 DNA solution + 1 Dye |
5 | SpeI (new) vs HJ's Ligase | 5 DNA solution + 1 Dye |
6 | SpeI (new) vs iGEM ligase | 5 DNA solution + 1 Dye |
7 | SpeI (old)(from last night) | 1 DNA solution + 1 Dye + 4 H2O |
8 | 1kb Ladder | 0.25 Ladder + 4.75 H2O + 1 Dye |
No observable ligation occurring 30 minutes with both ligases.
Objective: Prepare DNA for sequencing
Method: Need 20µL of DNA at 100ng/µL for each reaction. Need 20µL of 10µM primer for every 5 reactions.
Plasmids that need to be sequenced:
Name | Concentration (ng/µL) | Dilution Factor | Final Concentration (ng/µL) |
A8-CFP complete | 1395 | 1/5 | 280 |
B4-pSB NEYFP | 1105 | 1/5 | 240 |
B5-pSB CEYFP | 1055 | 1/5 | 210 |
B6-CFP complete | 1285 | 1/5 | 260 |
D7-xylE | 1820 | 1/10 | 182 |
D8-xylE | 420 | 1/2 | 210 |
E1-NEYFP | 235 | 1/2 | 117.5 |
E2-NEYFP | 2980 | 1/10 | 300 |
E3-Fusion CEYFP | 255 | 1/2 | 122.5 |
E4-Fusion CEYFP | 490 | 1/2 | 245 |
E5-Fusion CEYFP | 335 | 1/2 | 335 |
E6-CEYFP | 1605 | 1/10 | 160 |
E7-CEYFP | 1930 | 1/10 | 190 |
G4-pSB CEYFP | 340 | 1/4 | 85 |
G5-CFP complete | 355 | 1.4 | 89 |
Total of 15 primers -> 30 reactions
60µL of each VF2 & VR primers (10µL) -> send 65µL
(in lab: JV, HB)
Objective:Run Agarose gel of overnight samples from June 16/10 to test T4 DNA ligase
Method:Analyze ligation on a 1% TAE agarose gel.
Lane | Sample | Load (µL) |
1 | No Enzyme Control (from June 15) | 2 DNA solution + 2 Dye + 8 H2O |
2 | EcoRI + SpeI (old)(from June 15) | 2 DNA solution + 2 Dye + 8 H2O |
3 | EcoRI + SpeI (new) vs HJ's Ligase | 10 DNA solution + 2 Dye |
4 | EcoRI + SpeI (new) vs iGEM ligase | 10 DNA solution + 2 Dye |
5 | SpeI (new) vs HJ's Ligase | 10 DNA solution + 2 Dye |
6 | SpeI (new) vs iGEM ligase | 10 DNA solution + 2 Dye |
7 | SpeI (old)(from last night) | 10 DNA solution + 2 Dye |
8 | 1kb Ladder | 0.5 Ladder + 9.5 H2O + 2 Dye |
Ran for 80 minutes at 100V.
Results:
IMAGE TO COME!!!!
(in lab: JS, AS, KG)
Objective:Do PCR to observe ligations
Method:
Master Mix | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | 10.8 | 59.4 |
DNA | 2 | ------- |
5X Phusion Buffer | 4 | 22 |
10µM dNTP's | .25 | 8 |
Forward Primer | 1 | 5.5 |
Reverse Primer | 1 | 5.5 |
Polymerase | 0.2 | 1.1 |
Reaction Mixtures:
(in lab: JV)
Objective:Observe the ligations from June 17/10 that were amplified using PCR.
Method:Observe using 1% TAE agarose gel electrophoresis. Ran the gel for 80 minutes at 100V.
Lane | Sample | Volume (µL) |
8 | 1kb Ladder | 2 Ladder + 8 H2O + 2 Dye |
2 | control no DNA | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
3 | control no reverse primer | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
4 | control no forward primer | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
5 | HJ single | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
6 | iGEM single | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
7 | HJ double | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
8 | HJ double | 20 PCR reaction + 4 6X Dye and used 12 of this solution |
Results:IMAGE TO COME!!!!
(in lab: JV)
Objective:Create more pSB1A3 plasmid.
Method:Run a PCR of the plasmid.
Master Mix | Volume/tube (µL) |
MilliQ H2O | 10.8 |
DNA | 2 |
5X Phusion Buffer | 4 |
10µM dNTP's | .25 |
Forward Primer | 1 | DIlute 10X -> 0.1 + 0.9 MilliQ H2O
Reverse Primer | 1 | DIlute 10X -> 0.1 + 0.9 MilliQ H2O
Polymerase | 0.2 |
Use ligtest setting. WIll run 36 cycles.
Use 3 controls:
Results:PCR samples were run on a gel (1% 1XTAE) with the PCR samples from June 22/10. This gel was run on June 22/10
(in lab: JV, HS)
Objective:To overexpress pLacI-sRBS-lumazine synthase-dt in DH5α cells.
Method:Incubate cells in 500mL LB w/Tet. At OD 0.6 (600λ) induce overexpression of lumazine synthase with IPTG (1µM). Take To, T1, T2, T3 readings after induced with IPTG.
Time | OD F1 (600λ) | OD E10 (600λ) |
0 | 0.078 | 0.072 |
60 | 0.122 | 0.110 |
90 | 0.128 | 0.125 |
120 | 0.140 | 0.138 |
150 | 0.149 | 0.157 |
180 | 0.169 | 0.183 |
210 | 0.205 | 0.230 |
240 | 0.243 | 0.276 |
270 | 0.278 | 0.323 |
300 | 0.317 | 0.398 |
330 | 0.394 | 0.428 |
360 | 0.394 | 0.480 |
390 | 0.441 | 0.553 |
400 | ---- | 0.594 (induced) |
410 | 0.502 | ----- |
450 | 0.580 (induced) | 0.736 |
510 | 0.804 | 1.005 |
570 | 1.065 | 1.066 |
630 | 1.053 | 1.043 |
∞ | 0.055 (1/10 dilution) | 0.078 (1/10 dilution) |
Objective:PCR amplify the following biobricks:
Method:
Master Mix | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | ---- | 89.6 |
DNA | 2 | ------- |
5X Phusion Buffer | 4 | 24 |
10µM dNTP's | .4 | 2.4 |
Forward Primer | 0.2 | 1.2 |
Reverse Primer | 0.2 | 1.2 |
Polymerase | 0.2 | 1.2 |
1.2% Agarose gel electrophoresis (1X TAE), used to analyze the above PCR's. 25mL of agarose solution was used in the small gel rig.
Lane | Sample | Volume (µL) |
1 | pSB1A3 | 1 PCR sample + 2 Dye + 9 H2O |
2 | no DNA | 1 PCR sample + 2 Dye + 9 H2O |
3 | no reverse primer | 1 PCR sample + 2 Dye + 9 H2O |
4 | no forward primer | 1 PCR sample + 2 Dye + 9 H2O |
5 | 1kb ladder | 0.5 ladder + 2 dye + 9.5 Milli-Q H2O |
6 | mms6 | 1 PCR sample + 2 Dye + 9 H2O |
7 | dt (23L) | 1 PCR sample + 2 Dye + 9 H2O |
8 | dt (6K) | 1 PCR sample + 2 Dye + 9 H2O |
9 | dt (C1) | 1 PCR sample + 2 Dye + 9 H2O |
10 | no DNA | 1 PCR sample + 2 Dye + 9 H2O |
Ran the gel for 40 minutes at 100V.
(in lab: KG, AS)
Objective:To run a PCR of:
so that we have more DNA for restrictions and ligations.
Method:
Master Mix 1 | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | 10.8 | 226.8 |
DNA | 2 | 4 |
5X Phusion HF Buffer | 4 | 84 |
10µM dNTP's | 1 | 21 |
Forward Primer (VF2) | 1 | 21 |
Reverse Primer (VR) | 1 | 21 |
Fimnzzymes polymerase | 0.2 | 4.2 |
Master Mix 2 | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | 10.8 | 226.8 |
DNA | 2 | 4 |
5X Econo Taq Buffer | 4 | 84 |
10µM dNTP's | 1 | 21 |
Forward Primer (VF2) | 1 | 21 |
Reverse Primer (VR) | 1 | 21 |
Imitrages polymerase (Econo Taq) | 0.2 | 4.2 |
Master Mix for pSB1A3 | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | 10.8 | 31 |
DNA | 1 | 20 |
5X Phusion Buffer | 4 | 10 |
10µM dNTP's | 1 | 2.5 |
Primer (SB-prep-2Ea) | 1 | 2.5 |
Primer (SB-prep-3P) | 1 | 2.5 |
Fimnzzymes polymerase | 0.2 | 0.5 |
Master Mix for pSB1A3 | Volume/tube (µL) |
MilliQ H2O | 10.8 |
DNA | 2 |
5X Econo Taq Buffer | 4 |
10µM dNTP's | 1 |
Primer (SB-prep-2Ea) | 1 |
Primer (SB-prep-3P) | 1 |
Imitrages polymerase | 0.2 |
(in lab: JV, HS)
Objective:: To run the pcr products on an agarose gel.
Method: Run on 1% agarose gel using the large gel rig.
Lane | Sample | Load (µL) |
1 | 1kb DNA Ladder | 0.5 Ladder + 2 Dye + 9 H2O |
2 | Phu-dt-J9 | 5 PCR sample + 2 Dye + 3 H2O |
3 | Phu-dt-J10 | 5 PCR sample + 2 Dye + 3 H2O |
4 | Phu-dt-C1 | 5 PCR sample + 2 Dye + 3 H2O |
5 | Fus-mms6-B9 | 5 PCR sample + 2 Dye + 3 H2O |
6 | Fus-mms6-F10 | 5 PCR sample + 2 Dye + 3 H2O |
7 | Fus-rbs-xylE-I1 | 5 PCR sample + 2 Dye + 3 H2O |
8 | Fus-rbs-xylE-I2 | 5 PCR sample + 2 Dye + 3 H2O |
9 | Fus-xylE-D7 | 5 PCR sample + 2 Dye + 3 H2O |
10 | Fus-xylE-D8 | 5 PCR sample + 2 Dye + 3 H2O |
11 | Fus-pLacI-sRBS-lum-dt I8 | 5 PCR sample + 2 Dye + 3 H2O |
12 | Fus-pLacI-sRBS-lum-dt I9 | 5 PCR sample + 2 Dye + 3 H2O |
13 | Fus-sRBS-lum-dt A1 | 5 PCR sample + 2 Dye + 3 H2O |
14 | Phu-sRBS-lum-dt A2 | 5 PCR sample + 2 Dye + 3 H2O |
15 | Phu-sRBS-lum-dt B7 | 5 PCR sample + 2 Dye + 3 H2O |
16 | Phu-sRBS-lum-dt B8 | 5 PCR sample + 2 Dye + 3 H2O |
17 | Phu-sRBS-lum-dt G2 | 5 PCR sample + 2 Dye + 3 H2O |
18 | Phu-sRBS-lum-dt G3 | 5 PCR sample + 2 Dye + 3 H2O |
19 | Phu-pLacI A9 | 5 PCR sample + 2 Dye + 3 H2O |
20 | Phu-pLacI D1 | 5 PCR sample + 2 Dye + 3 H2O |
21 | dt J9 | 5 PCR sample + 2 Dye + 3 H2O |
22 | dt J10 | 5 PCR sample + 2 Dye + 3 H2O |
23 | dt C1 | 5 PCR sample + 2 Dye + 3 H2O |
24 | mms6 B9 | 5 PCR sample + 2 Dye + 3 H2O |
25 | mms6 F10 | 5 PCR sample + 2 Dye + 3 H2O |
26 | rbs-xylE I1 | 5 PCR sample + 2 Dye + 3 H2O |
27 | rbs-xylE I2 | 5 PCR sample + 2 Dye + 3 H2O |
28 | xylE D7 | 5 PCR sample + 2 Dye + 3 H2O |
29 | xylE D8 | 5 PCR sample + 2 Dye + 3 H2O |
30 | pLacI-sRBS-lum-dt I8 | 5 PCR sample + 2 Dye + 3 H2O |
31 | pLacI-sRBS-lum-dt I9 | 5 PCR sample + 2 Dye + 3 H2O |
32 | sRBS-lum-dt A1 | 1 PCR sample + 2 Dye + 9 H2O |
33 | sRBS-lum-dt A2 | 1 PCR sample + 2 Dye + 9 H2O |
34 | sRBS-lum-dt B7 | 1 PCR sample + 2 Dye + 9 H2O |
35 | sRBS-lum-dt B8 | 1 PCR sample + 2 Dye + 9 H2O |
36 | sRBS-lum-dt G2 | 1 PCR sample + 2 Dye + 9 H2O |
37 | sRBS-lum-dt G3 | 1 PCR sample + 2 Dye + 9 H2O |
38 | sRBS-lum-dt A9 | 1 PCR sample + 2 Dye + 9 H2O |
39 | sRBS-lum-dt D1 | 0.5 ladder + 2 dye + 9.5 Milli-Q H2O |
40 | pSB1A3 control | 5 PCR sample + 2 Dye + 3 H2O |
41 | pSB1A3 | 5 PCR sample + 2 Dye + 3 H2O |
42 | pSB1A3 control | 5 PCR sample + 2 Dye + 3 H2O |
43 | pSB1A3 | 5 PCR sample + 2 Dye + 3 H2O |
Ran gel for 45 minutes at 100V and stained in EtBr for 20 minutes and de-stained for 10 minutes.
Results:: Picture to come!!!!!!!!!
Objective: Adam put in overnight cultures on June 22. I want to extract the plasmid DNA.
Method:
Results: Will view plasmid DNA extracted, on a 1% agarose gel (1X TAE) run at 100V for an hour.
Lane | Sample | Load (µL) |
1 | 1kb Ladder | 0.5 Ladder + 2 Dye (6X) + 9.5 Milli-Q H2O |
2 | CFP complete 2010 box C8 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
3 | Fusion CEYFP 2007 box H5 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
4 | NEYFP 2007 box J6 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
5 | Fusion CEYFP 2007 box J5 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
6 | CFP complete 2010 box A10 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
7 | pSB-NEYFP 2010 box B6 (C1?) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
8 | Fusion CEYFP 2007 box I5 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
9 | xylE 2007 box B9 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
10 | CEYFP 2007 box G6 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
11 | CFP complete 2010 box A6 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
12 | pSB-CEYFP 2010 box C5 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
13 | pSB-NEYFP 2010 box B6 (C1?) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
14 | pSB-CEYFP 2010 BOX C3 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
15 | xylE 2007 box C4 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
16 | CEYFP 2007 box H6 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
17 | NEYFP 2007 box J6 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
Picture to come!!!!!!!!!
(in lab: JV, HS, AV)
Objective:: To run a PCR of biobrick parts from out working plasmid box.
Method:
Master Mix | Volume/tube (µL) | Total Volume (µL) |
MilliQ H2O | 12.8 | 448 |
DNA | 1 | ------- |
5X Phusion Buffer | 4 | 140 |
10µM dNTP's | 1 | 35 |
Forward Primer | 0.5 | 17.5 |
Reverse Primer | 0.5 | 17.5 |
Polymerase | 0.2 | 7 |
Ran for 25 cycles on lig25 setting.
Objective:: Restriction Digest, Run on agarose gel, and ligate PCR products from June 23/10.
Method:
Six tubes for restriction digest:
Part 1 | Part 2 |
Lane 8 (I2) | Lane 4 (C1) |
Lane 19 (A9) | Lane 8 (I2) |
Lane 19 (A9) | Lane 8 (I2) |
Restriction Digest Reaction Mixture:
Ingredient | Volume (µL) |
Plasmid DNA | 2 |
Enzyme | 0.5 |
Buffer | 2 |
Milli-Q H2O | 15.5 |
Incubate at 37oC for 1 hour. Then heat kill the enzymes at 80oC for 20 minutes.
2% Agarose gel electrophoresis:
Lane | Sample | Load (µL) |
1 | 50bp Ladder | 1 Ladder + 2 Dye (6X) + 5 Milli-Q H2O |
2 | Fus-RBS-xylE (I2); Lane 8 | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
3 | Fus-RBS-xylE (I2) restricted; Lane 8 | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
4 | Phu-dT (C1); Lane 4 | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
5 | Phu-dT (C1) restricted; Lane 4 | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
6 | Phu-pLacI (A9); Lane 19(1) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
7 | Phu-pLacI (A9) restricted; Lane 19(1) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
8 | Phu-pLacI (A9); Lane 19(2) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
9 | Phu-pLacI (A9) restricted; Lane 19(2) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
10 | Fus-RBS-xylE (I2); Lane 8(1) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
11 | Fus-RBS-xylE (I2) restricted; Lane 8(1) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
12 | Fus-RBS-xylE (I2); Lane 8(2) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
13 | Fus-RBS-xylE (I2) restricted; Lane 8(2) | 5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O |
Ran gel at 100V for 60 min and stained in EtBr for 15 min.
Picture to come!!!!!!!!!
Objective:: Run 1% agarose gel of PCR samples from June 24/10.
Lane | Sample | Load (µL) |
1 | A4-pBAD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
2 | A4-pBAD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
3 | A6-SRBS | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
4 | A7-SRBS | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
5 | A8-CFP Complete | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
6 | A10-SRBS | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
7 | B1-EYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
8 | B2-N-term tag | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
9 | B4-pSB-NEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
10 | B5-pSB-NEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
11 | B6-CFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
12 | B10-pBAD-TetR | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
13 | D3 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
14 | D4-C-term tag | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
15 | D5-C-term tag | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
16 | D6-PLacI | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
17 | E2-NEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
18 | E6-CEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
19 | E7-CEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
20 | 1kb ladder | 0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O |
21 | E8-EYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
22 | E9-EYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
23 | E10-ECFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
24 | F1-ECFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
25 | F2-ECFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
26 | F3-ECFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
27 | F4-pBAD-TetR | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
28 | F5-pBAD-TetR | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
29 | G1-EYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
30 | G4-pSB-CEYFP | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
31 | G6-pBAD(1) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
32 | G7-pBAD(2) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
33 | G8-N-term tag | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
33 | G9-Lumazine | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
33 | 1kb ladder | 0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O |
Picture to come!!!!!!!!!
(in lab: JV, HS, AV, HB)
Objective:: To restrict, run 1% agarose gel, and ligate all mms6, lumazine, xylE into pET28(a) for overexpression tests.
Method:
1) Restrict all parts and pET28(a) with NotI
2) Run on agarose gel (1%)
3) Ligate parts into pET28(a)
Parts:
Restrictions:
Protocol:
Agarose Gel:
Lane | Sample | Load (µL) |
1 | 1kb ladder | 0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O |
2 | Lumazine A3 RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
3 | Lumazine A3 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
4 | Mms6 I10 RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
5 | Mms6 I10 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
6 | Mms6 B9 RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
7 | Mms6 B9 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
8 | xylE D8 RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
9 | xylE D8 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
10 | xylE D7 RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
11 | xylE D7 | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
12 | Lumazine G9 RD (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
13 | Lumazine G9 (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
14 | xylE B9 RD (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
15 | xylE B9 (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
16 | xylE C4 RD (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
17 | xylE C4 (p) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
18 | pET28(a) RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
19 | pET28(a) RD | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
20 | pET28(a) | 5 DNA + 2 Dye (6X) + 5 Milli-Q H2O |
Ran gel at 100V for 90 minutes
Results:pET28(a) bands are all smeared, and are not useful from inserting parts.
Picture to come!!!!!!!!!
(in lab: JV)
Objective:: Isolate plasmid DNA from DH5α
Method:Used Kothe Maxiprep protocol.
Cell Pellet Weights:
Results: