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Team:Alberta/Notebook/Assembly Method
From 2010.igem.org
25-05-2010
Discussed an assembly method. Will start with a polyA tail Anchor oligo which will connect to a polyT tail which is bound to the bead. The anchor will consist of a primer region, a BsaI cut site, a buffer region, and finally, an A prime or B prime end. A byte with an appropriate end can be added to this anchor, and another byte can be added onto that, etc... The last thing to be added to the construct is a Terminator byte consisting of a buffer region, BsaI cut site, primer region and a polyT tail. This second primer sequence must be as uncomplimentary as possible to the anchor's primer site so as to minimize primers binding to each other when PCRing the assembled construct. The primers melting point must be around 65 C. After the construct is assembled on the bead, it is heated off and the polyA tail of the anchor binds to the polyT tail of the terminator and a plasmid is born.
We will purchase NEB oligo dT magnetic and cellulose beads which have a polyT tail (5' to 3') 25 nucleotides long. They are meant for mRNA isolation, but should work fine for our purposes. Used the IDT Analyzer software to determine what length of polyA tail is required for our anchor to have a melting temperature of 30 degrees Celsius (decided to change this melting temp later). This melting temp is sensitive to Na+ and Mg++ concentrations, so referred to ligase buffer and elution buffer for these concentrations.
The BsaI A end should look like this:
Designed an anchor with a polyA10 tail with a primer (Tm=65.1 C) region and the BsaI cut site with an A' end (ACCC).
26-05-2010
Designed new anchors, ordered oligos. Designed new anchors with larger polyA tail (and without hairpins, or self dimerization).
27-05-2010
No binding capacity specified in the manual for the beads in terms of mols or mass of DNA or beads. Binding capacity is given in terms of cells (because the beads are meant for mRNA isolation from cells). Will have to quantify binding capacity later.
Derivation of mole matching equation: ssDNA is ~330 grams/(mol*bp) moles = [concentration X volume] / [(330grams/mol X bp) X length] moles1 = moles2 c1v1/[(330g/mol X bp) X l1] = c2v2/[(330g/mol X bp) X l2] v1 = v2(c2 X l1/ c1 X l2)
Ordered new primers for anchor: GCG CGC CCG GTC TCA TGG GTC ACC CTC CC GGG AGG GTG ACC CAT GAG ACC GGC GCG C[A]12
07-06-2010
Made Wash Buffer, Elution Buffer and Low Salt Buffer for the mRNA Isolation Kit.
08-06-2010
Produce a modified protocol for Anchor binding to cellulose beads.
Protocol Preparation:
- Allow everything to come to room temperature.
- Spin 2000 to 5000g of beads in microcentrifuge for 10 seconds.
- Remove supernatant without disturbing the beads.
- Add 200μl of Wash Buffer to beads and agitate.
- Centrifuge for 10 seconds and remove supernatant.
- Pre-warm Elution Buffer in a 70oC water bath.
Isolation Procedure:
- Apply DNA Anchor to cellulose beads, agitate and let it stand at room temperature for 5 minutes.
- Microcentrifuge for 10 seconds.
- Pipette supernatant back into original microcentrifuge.
- [Add 4μl Wash Buffer to beads and agitate to resuspend. Transfer beads and wash buffer to column reservoir of spin column. Let it stand at room temperature for 2 minutes while agitating. Microcentrifuge for 10 seconds.] X3
- Add 400μl Low Salt Buffer, resuspend by agitation for 2 seconds and microcentrifuge for 10 seconds.
- Transfer and place spin column reservoir in a clean microcentrifuge tube.
- [Add 200μl pre-warmed Elution Buffer to column reservoir. Agitate to resuspend beads and let it stand for 2 minutes. Microcentrifuge for 10 seconds] X2
- Place eluent on ice.
09-06-2010
Calculated the mass of beads to determine the range of mg RNA could be isolated. 0.06g beads = 0.0599g; therefore, 0.06g can isolated 0.1 to 1 mg RNA.
Calculated the minimal and maximal rpm for 2000 to 5000g of beads using the following formula: a = 4(pi)2r(rpm)2 / 602 So, when centrifuging the beads, the rpm must stay between 4700 to 7400 rpm. In the modified mRNA procedure, the centrifuge is set to 5500 rpm.
10-06-2010
Attempted to find binding capacity (in moles per mass of beads) by binding known masses of anchor to the bead, following the protocol 08-06-10, then quantifying the mass of DNA recovered off of the beads after eluting the DNA off of the bead. The cellulose beads settle very quickly without agitation. Measuring precise volumes of the beads was done by pipetting the cellulose bead slush into pcr tubes that were marked at 16 uL. To quantify the masses of DNA that: did not bind to the bead, came through the washes, and were eluted from the beads, the nano-drop 1000 spectrophotometer was used. The results showed that most of the anchor did not bind to the cellulose beads, and a very small amount of DNA was bound and eluted off of the beads. Possible reasons could be that the anchor was not binding, or there was too much anchor relative to the beads. A gel was also done. It showed that there was a lot of anchor that did not bind, and no anchor band was visible in the elution lane (which was expected based on the nanodrop reading).
11-06-2010
Repeated yesterday's experiment, but with a much larger amount of DNA. 1500 ng of anchor DNA to 16 uL of cellulose bead slush. There were no significant nanodrop readings except for the measurement of the amount of DNA that did not bind to the beads. Based on the nanodrop reading, over 1100 ng of DNA did not bind to the beads. The rest must have been washed away in the wash. Perhaps the melting temperature of the polyA12 tail is too low. We'll order one with a polyA18 tail and repeat the experiment.
14-06-2010
-Attempted to bind various masses of anchor to 16 uL of beads. Used 500, 1000, and 1500 ng of beads. Followed the modified mRNA isolation protocol created last week 08-06-10. Based on nanodrop readings, we recovered 560 ng from 1500 ng of initial DNA, 63 ng of 1000 ng of initial DNA, and 244 ng of 500 ng of DNA. Strange result of the 1000 ng of DNA, but a good sign that something appears to be binding to the cellulose beads. -To attempt to find the amount of anchor that will saturate the beads binding capacity, we used 1, 1.5, 2, and 2.5 ug of anchor and attempted to bind them to 16 uL of cellulose beads. Nanodrop readings multiplied by volume gave 600, 763, 1102, and 1730 ng of DNA not binding to the bead. What bound to the bead was 248, 1935, 4032, and 2470 ng of DNA. Obviously, something has caused the DNA readings to imply that more DNA was recovered than was put in initially. Later we found out that the readings are extremely sensitive to the small amount of residual washing solutions left over, causing the blanking process of the nanodrop to not accurately represent what solution the DNA was in.
15-06-2010
-The cellulose mRNA isolation kit came with a small amount of columns enough for only 8 trials. More were ordered. Will use magnetic beads in the meantime. -Created modified magnetic bead protocol based on the mRNA isolation protocol 08-06-10. Enough beads present in kit for 100 trials. -Magnetic Bead Trial #1: attempt to bind 100, 500, 1000, and 2500 ng of polyA12 anchor to 20 uL of polyT magnetic beads. Followed the modified magnetic bead protocol and eluted in 50 uL of elution buffer in a 50 C water bath. When nanodropping, apply magnet in case a small amount of magnetic particles were caught in the elution. Results: 80, 250, 305, and 460 ng of DNA were recovered from the beads after applying 100, 500, 1000 and 2500 ng of DNA initially. The magnetic beads appear to be more efficient at lower masses of DNA being bound. This appears encouraging, however, the nanodrop readings multiplied by the solution volume for the amount of DNA that did not bind initially were 335, 785, 970, and 2140 ng of DNA. The amount of DNA that did not bind and the amount that did does not add up to the amount of DNA added. This could be due to the residual solution left behind after washes. Will redo the experiment with less concentrated anchor and careful washes.
16-06-2010
-Magnetic Bead Trial 2: attempt to bind 500, 1000, 2500, and 5000 ng of anchor to 20 uL of beads. The mass of the DNA which didn't bind exceeded the mass of what was intitially mixed with the beads. This is obviously an error in measurement (caused by what!). The amount of DNA recovered was 0, 90, 70, and 250 ng of DNA. Looks like nothing bound to the beads. Possible cause of no DNA binding to bead: very low concentration of anchor meant a large reaction volume which would lead to a higher reaction time. Next time, use higher concentration anchor and increase binding time to an hour rather than 15 minutes (the mRNA isolation procedure calls for only 10 minutes).
17-06-10
Testing Absorbance of Water and Beads with Applied Magnet
- Allow beads to sit in Binding Buffer, applied magnet, discard supernatent and add MilliQ H2o.
- Remove and keep supernatent (Mimics what didn't bind to beads) and add more MilliQ to beads.
- Nanodrop samples.
- Results: Traces of Lysis Buffer skews readings on Nanodrop. For next experiment use Washing Buffer instead of Lysis Buffer.
Magnetic Bead Trial #3:
- Used approximately 250, 500, 1000, 2000 ng of anchor to 20μL of beads in modified mRNA Isolation Protocol. Nanodrop samples during different stages of procedure.
- Results: Saturation point was reached before 486 ng for 100μL beads. Next experiment, test higher volumes of anchor with more volume of beads.
Magnetic Beads Trial #4:
- Used 200, 400, 800 and 1600 ng of anchor to 40μL beads using the same protocol in the previous trial.
Anchor Melting Points Test:
- Saturate 40μL beads with 5000 ng of anchor. Wash once with Washing Buffer before adding MilliQ H2o and ligase buffer. Test samples at room temperature (22oC), 30oC, 40oC and 50oC.
- Nanodrop samples.
- Results: Readings indicate there is DNA present even after wash. Next experiment, wash twice instead of once and measure second wash to confirm whether DNA is present or not. Test both types of anchors and allow annealing of anchors to polydT of beads at a slower rate.
23-06-10
Made new buffers for Magnetic Beads.
25-06-10
Anchor Melting Temperature Test:
- Tested polyA12 and polyA18 anchors in ligase buffer and elution buffer following modified mRNA Isolation Protocol
- Place in temperatures of 22oC, 25oC, 30oC, 35oC, 40oC, 45oC, 50oC, 55oC, 60oC, 65oC, 70oC, 75oC and 85oC.
28-06-10
Anchor Melting Temperature Test #1:
- Nanodrop samples in ligase buffer and elution buffer on 25-06-10 to plot a graph to determine melting point of anchors. *Contact Eric for graph!!*
30-06-10
Anchor Melting Temperature Test #2:
- Redo experiment at higher temperatures: 30oC, 35oC, 40oC, 45oC, 50oC, 55oC, 60oC, 65oC, 70oC, 75oC, 85oC, 90oC and 95oC.
- Nanodrop samples to plot a graph to determine melting point of anchors.
- Results: Compared graphs produced today and on 28-06-10 and used IDT to determined melting point of both anchors.
- polyA18 Tm=51.3oC
- polyA12 Tm=35.2oC
05-07-10
Bromophenol Blue affects on Ligation Test:
- Digested 1μg of plasmid and purified to get 1kb and 2kb fragments.
- Dissolve 0.1g of BPB in 40mL Tris-EDTA and 10mL 70% Ethanol. Made 1/2, 1/8 and 1/32 dilutions of BPB.
- Ligate cut plasmid with BPB overnight at room temperature.
06-07-10
Bromophenol Blue affects on Ligation Test:
- Take the ligations made on 05-07-10 and run it on a gel.
- Results: BPB inhibits ligation. Next experiment, try again using Cresol Red instead of Bromophenol Blue.
08-07-10
Formulate an equation to calculate the amount of anchor (in moles) to Byte.
VAnc = VKan (CKan/CAnc) (LAnc/LKan)
10-07-10
Testing Digestion and Ligation of Anchor to Byte:
- Digested Kan Bsa AB with BsaI, PCR Purify and ligate to anchor.
<---10.07.10 Eric--->
- Results: Anchor ligated to digested plasmid.
13-07-10
Testing Anchor Binding to Beads:
- Calculated volumes required for 20X and 50X anchor to bind to cellulose and magnetic beads. Ligated Byte to anchor, used mRNA Isolation Protocol to bind anchor with Byte to beads.
- Will run a gel of flowthrough, washes and elution for cellulose and magnetic beads.
14-07-10
Testing Anchor Binding to Beads: <---14.07.10 Eric--->
- Results: High amount of flowthrough, but some binding to magnetic beads.
<---14.07.10 Oscar--->
- No anchor binding to cellulose beads.
- Re-do Anchor Binding to Beads, but use only 20X anchor and PCR Purify to decrease extra anchor not bound to the beads. More beads were used.
<---14.07.10 Eric (Cellulose)---> <---14.07.10 Oscar (Magnetic)--->
- Results: Anchor binding to Magnetic Beads worked while there is no anchor binding to cellulose beads.
16-07-2010
Gel Purified digestions of Kan AB and BA Bytes.
Solution Phase Assembly (Preparations)
Digested 10μg of pSB1C3 with Amp Resistance using BsaI. This digested DNA will be used to test: 1) whether acyl-NTPs will specifically incorporate to the cut ends of DNA by Klenow Polymerase and 2) if the acyl-NTPs block the ligation reaction between the vector (pSB1C3) and the insert (Amp Resistance). Two different minipreps of pSB1C3 with AmpR were used for the digestions.
Digestion #1 Protocol:
- 28.3μL pSB1C3 with AmpR (176.5ng/μL nanodrop concentration)
- 60.7μL MilliQ H20
- 10.0μL 10X NEBuffer 4
- 1.0μL BsaI
- 100μL Total
Digestion #2 Protocol:
- 28.7μL pSB1C3 with AmpR (174.1ng/μL nanodrop concentration)
- 60.3μL MilliQ H20
- 10.0μL 10X NEBuffer 4
- 1.0μL BsaI
- 100μL Total
Both were incubated at 50oC for 1 hour, were PCR Purified using the Qiagen PCR Purification Kit and ran on a 1.0% agarose gel to check for completion.
Gel Electrophoresis:
<---16.07.10 Anh--->
Only lanes 6 to 8 were used. Lane 6 contains the kb+ ladder, while Lanes 7 and 8 contains the digested and purified pSB1C3 with AmpR fragments. Overall, the digestion went to completion.
19-07-2010
Solution Phase Assembly (Pre-test #1)
Used the digested pSB1C3 with AmpR that were prepared on 16-07-2010.
Protocol:
- Prepared acyl-NTPs by adding 50μL of MilliQ H20 to each powdered acyl-NTP (acyl-ATP, acyl-CTP, acyl-GTP and acyl-TTP). Then, add equal volume amounts of each acyl-NTP solution in an eppendorf tube to produce the complete mix to be used in the pre-test.
- Set-up 4 tubes with 2μL of 0.5M EDTA. The EDTA will be used to inhibit Klenow Polymerase's enzyme activity.
- Prepare Blocking Solution by adding: 85μL cut pSB1C3 and AmpR, 4μL complete acyl-NTP mix and 10μL Klenow Buffer.
- Transfer 20μL of Blocking Solution in one tube with 0.5M EDTA. This is the control of the experiment. It was placed in a 75oC water bath for 20 minutes to heat inactivate Klenow.
- Add 5μL Klenow Polymerase to the remaining Blocking Solution and incubate at 37oC.
- 20μL of the Blocking Solution was transferred to the remaining tubes with 0.5M EDTA after 5, 10 and 20 minutes incubation at 37oC. These tubes were also heat inactivated at 75oC for 20 minutes.
- All tubes were PCR Purified using the Qiagen PCR Purification Kit.
- Ligation of digested pSB1C3 and AmpR was done after purification.
- 16μL digested pSB1C3 and AmpR with acyl-NTP.
- 2μL of 5X ligase buffer
- 2μL of T4 ligase
- Ligations were incubated at room temperature for 1 hour.
20-07-2010
Bytes Ligating Test with Anchor #1:
- Calculated volumes of anchor to Kan AB and BA Bytes, did a ligation and used modified mRNA Isolation protocol.
- Ran a gel, which resulted in lots of smears. Next experiment, re-do test using gel-purified Bytes.
Solution Phase Assembly (Pre-test #1)
Ran the samples done on 19-07-2010 on a 1.0% agarose gel.
Gel Electrophoresis:
<---20.07.10 Anh--->
Lane 1 is the kb+ ladder. There are two controls: Lane 2 contains the cut pSB1C3 and Amp Resistance, and Lane 3 contains Blocking Solution but no Klenow Polymerase. Lanes 4 to 6 contains Blocking Solution with Klenow Polymerase but vary in incubation times for Klenow's enzyme reactivity. Lane 4 incubated for 5 minutes, Lane 5 incubated for 10 minutes and Lane 6 incubated for 20 minutes. Results: Lane 3 proceeded to ligate the DNA while Lanes 4 to 6 did not. Therefore, 5 minutes is the minimum amount of time to allow Klenow Polymerase to incorporate acyl-NTPs at the ends of digested pSB1C3 and AmpR, which prevents ligase's function to ligate the DNA.
21-07-2010
Bytes Ligating Test with Anchor #2:
- Digested Kan B/A plasmid with BsaI.
Solution Phase Assembly (Pre-test #2)
The next objective is to see if: 1) Klenow Polymerase can function efficiently in ligase buffer instead of Klenow Buffer to see if the PCR Purification step can be skipped, 2) to check whether acyl-NTPs are the one responsible for inhibiting ligase's activity, and 3) does 1X SDS enhance the visual of gel bands in 1.0% agarose gels.
Digestion of Kan Bsa B/A is needed before doing the experiment.
Digestion Protocol:
- 19.3μL Kan B/A Bsa (258.7ng/μL nanodrop concentration)
- 2.5μL 10X NEBuffer 4
- 2.2μL MilliQ H20
- 1.0μL Bsa I
Incubate at 50oC for 1 hour and PCR Clean-Up using Qiagen PCR Purification Kit.
Using the digested Kan Bsa B/A, proceed with the following protocols for Solution Phase Assembly (Pre-test #2): (Note that each tube contains a different solution mix with a total of 21μL, but they all have the same incubation times)
Protocol (Tube1):
- 16μL Kan B/A Bsa
- 2μL ligase buffer
- 2μL MilliQ H2O
Incubate at 37oC for 10 minutes.
Add 1μL MilliQ H2O.
Incubate at room temperature for 1 hour.
Protocol (Tube2):
- 16μL Kan B/A Bsa
- 2μL ligase buffer
- 1μL complete acyl-NTP mix
- 1μL MilliQ H2O
Incubate at 37oC for 10 minutes.
Add 1μL T4 ligase.
Incubate at room temperature for 1 hour.
Protocol (Tube3):
- 16μL Kan B/A Bsa
- 2μL ligase buffer
- 1μL Klenow Polymerase
- 1μL MilliQ H2O
Incubate at 37oC for 10 minutes.
Add 1μL T4 ligase.
Incubate at room temperature for 1 hour.
Protocol (Tube4):
- 16μL Kan B/A Bsa
- 2μL ligase buffer
- 2μL MilliQ H2O
Incubate at 37oC for 10 minutes.
Add 1μL T4 ligase.
Incubate at room temperature for 1 hour.
Protocol (Tube5):
- 16μL Kan B/A Bsa
- 2μL ligase buffer
- 1μL complete acyl-NTP mix
- 1μL Klenow Polymerase
Incubate at 37oC for 10 minutes.
Add 1μL T4 ligase.
Incubate at room temperature for 1 hour.
22-07-2010
Solution Phase Assembly (Pre-test #2)
Ran the samples done on 21-07-2010 on a 1.0% agarose gel.
Gel Electrophorsis:
<---22.07.10 Anh--->
Lane 1 and 2 contain the kb+ ladder and the cut Kan B/A Bsa control respectively. The following sequential lanes are: Tube 1, 2, 3, 4, 5, and Tube 1, 2, 3, 4, 5 with 1.0% SDS added with the loading dye. Results: In Lanes 8 to 12, the gel band visibility is more worse than the gel bands in Lanes 3 to 7, indicating that 1.0% SDS decreases visibility. One unexpected result is that the presence of Klenow appears to be inhibiting ligation (Tube 3 - Lane 5).
23-07-2010
Bytes Ligating Test with Anchor #2:
- Did a Gel Extraction of digests done on 21-07-10.
- Bind AB Byte to Anchor by ligation.
- Bind Anchor with AB Byte to Magnetic Beads by using modified mRNA Isolation protocol.
- Apply the second Byte by ligation. There are three types of ligations: ABanch + AB Byte + ligase, ABanch + BA Byte + ligase, and ABanch + BA Byte + no ligase.
- Flowthrough and elution samples were collected to be run on a gel.
Solution Phase Assembly (Pre-test #3)
Further tests are done to investigate whether Klenow Polymerase is inhibiting ligation. The experiment is set up to test different methods to physically remove the enzyme by PCR Purification or to deactivate its activity by heat inactivation. Taq Polymerase is also tested to see whether it would be a good substitute for Klenow Polymerase and its buffer compatibility with ligase buffer.
Digestion of Kan Bsa A/B is needed before doing the experiment.
Digestion Protocol:
- 21.6μL Kan B/A Bsa (231.6ng/μL nanodrop concentration)
- 3.0μL 10X NEBuffer 4
- 4.4μL MilliQ H20
- 1.0μL Bsa I
* The protocol was done in a master mix to produce 4 tubes so the remaining digested DNA could be used in future experiments.
Incubate at 50oC for 1 hour and PCR Clean-Up using Qiagen PCR Purification Kit.
Using the digested Kan Bsa A/B, proceed with the following protocols for Solution Phase Assembly (Pre-test #3):
Klenow Polymerase Protocol:
- 16μL Kan A/B Bsa
- 4μL 5X ligase buffer
- 1μL Klenow Polymerase
- * The protocol was done in a master mix to produce 3 tubes to test different methods after incubation.
- Incubated at 37oC for 10 minutes.
- Tube #1 was heat inactivated at 75oC for 20 minutes. Tube #2 was PCR Purified using the Qiagen PCR Purification Kit. And Tube #3 went directly to the ligation step (This is the control of the Klenow Experiment).
- Tube #1 and #3 had only 1μL T4 ligase added directly to the mix. Tube #2 has to transfer 16μL of digested Kan A/B Bsa into another eppendorf tube and had 4μL 5X ligase buffer and 1μL T4 ligase added to its mix.
- All tubes were incubated at room temperature for 1 hour.
Taq Polymerase Protocol #1:
- 16μL Kan A/B Bsa
- 4μL 5X ligase buffer
- 1μL complete acyl-NTP mix
- 1μL Taq Polymerase
Taq Polymerase Protocol #2:
- 16μL Kan A/B Bsa
- 2μL 10X Taq buffer
- 1μL complete acyl-NTP mix
- 1μL Taq Polymerase
Both Taq solutions were incubated at 75oC for 10 minutes. 1μL T4 ligase was added directly into the mix afterwards.
Gel Electrophoresis:
<---23.07.10 Anh--->
Results: The Taq experiment with ligase and acyl-NTPs did not inhibit completely (Lanes 6 and 7). Only Klenow experiment with PCR Purification undergo ligation. Therefore, it is possible that the presence of Klenow Polymerase inhibits the enzyme reaction of ligase.
24-07-10
Bytes Ligating Test with Anchor #2:
- Use only one third of each flowthrough and elution samples collected on 23-07-10 to be run on a 1.0% agarose gel.
<--24.07.10 Eric3-->
- Results: Significant excess of anchor present in the ABanch mix. PCR Purification is not efficient; therefore, use less than 20X anchor to AB Byte in next experiment.
