T Dna Primer Design
Projects Engaged
During this week and a half, I began trying to delete the BAR1 gene from each of the strains we are studying. We are pursuing this line of work in order to increase the efficacy of alpha-factor induced cell synchrony.
Results Achieved
I used PCR mediated gene disruption to add segments of DNA that are homologous to the segments of DNA that flank the BAR1 gene onto the ends of a gene that codes for kanamycin resistance.
First try:
Each reaction contained 30μL ddH20, 5μL 10X Taq buffer, 1μL 10mM dNTP mix, 5μL of each primer (10mM), 4μL 25mM MgCl2, 0.5μL BSA [10mg/mL (initial concentration)], and 0.5μL Taq Polymerase.
Reaction 1: 10ng of Kan plasmid from Grace Primers: 74mer UPTAG, 74mer DOWNTAG
Reaction 2: ~3μg genomic DNA Primers: ACT1 SHORT (F & R)
Reaction 3: No DNA Primers: ACT1 SHORT (F & R)
PCR cycle profile:
- 94⁰C – 4 min
- 94⁰C – 1 min
- 55⁰C – 1 min
- 72⁰C – 1:30 min
25 cycles - 72⁰C – 20 min
Lane 1: Reaction 1
Lane 2: Reaction 2
Lane 3: Reaction 3
Skipped lanes were not counted in lane numbering.
The target band is present (very faint) at ~2200bp in the first lane. I ran this gel quickly just to test the protocol. It looks like the PCR did work because it produced a band of the expected length. The yield is pretty low though. This would probably not be enough DNA to continue with.
Second try:
Because the yield was so low in the first PCR, I increased the plasmid template concentration to 100ng to try to increase the yield.
Each reaction contained 30μL ddH20, 5μL 10X Taq buffer, 1μL 10mM dNTP mix, 5μL of each primer (10mM), 4μL 25mM MgCl2, 0.5μL BSA [10mg/mL (initial concentration)], and 0.5μL Taq Polymerase.
Reaction 1: 100ng of Kan plasmid from Grace Primers: 74mer UPTAG, 74mer DOWNTAG
Reaction 2: 500ng Genomic DNA Primers: HSF1 SHORT (F & R)
Reaction 3: No DNA Primers: HSF1 SHORT (F & R)
PCR cycle profile:
- 94⁰C – 4 min
- 94⁰C – 1 min
- 55⁰C – 1 min
- 72⁰C – 1:30 min
35 cycles - 72⁰C – 10 min
Lane 1: Reaction 1
Lane 2: Reaction 2
Lane 3: Reaction 3
Skipped lanes were not counted in lane numbering.
More of the expected product was created in this PCR by increasing the number of cycles from 25 to 35 and increasing the amount of template 10X. The smear present in lane 1 is most likely from incomplete elongation. Because the band we’re looking for is about 2200bp long, the elongation step should be at least 2:12min (1min per kb). The negative control lane also has a pretty strong band even though it had no template. This probably means that the HSF1 short aliquots of primers are contaminated because the negative control using ACT1 short primers used in the previous series of PCRs worked well. The contamination could also be from using the BSA. Reaction 1 was cleaned with a 1:1 phenol:chloroform extraction, precipitated with 95% ethanol and 5μL 3M sodium acetate, and resuspended in 20μL ddH20 before it was run on the gel. This could have lead to a decrease in yield, but it shouldn’t have created the smear.
I cut out the ~2200 band and purified it with the QIAquick Gel Extraction Kit. The final concentration was 4.5ng/μL in 30μL of elution buffer. The curve shows a peak at 230nm and flatlines after that. The 260/280 is 1.93.
Third try:
To try to get rid of the smear from the previous PCR, I repeated the PCR with a longer elongation time.
Each reaction contained 30μL ddH20, 5μL 10X Taq buffer, 1μL 10mM dNTP mix, 5μL of each primer (10mM), 4μL 25mM MgCl2, 0.5μL BSA [10mg/mL (initial concentration)], and 0.5μL Taq Polymerase.
Reaction 1: ~120 ng of 1st round PCR product (all) Primers: 45mer UP_45, DOWN_45
Reaction 2: 100ng of Kan plasmid from Grace Primers: 74mer UPTAG, 74mer DOWNTAG
Reaction 3: Genomic DNA (1500ng) Primers: HSF1 SHORT (F & R)
Reaction 4: No DNA Primers: HSF1 SHORT (F & R)
PCR cycle profile:
- 94⁰C – 4 min
- 94⁰C – 1 min
- 55⁰C – 1 min
- 72⁰C – 3 min
35 cycles - 72⁰C – 20 min
Lane 1: Reaction 1
Lane 2: Reaction 2
Lane 3: Reaction 3
Lane 4: Reaction 4
Skipped lanes were not counted in numbering.
There wasn’t a band of the expected length in the Round 2 PCR which was disappointing, because there was enough template. Actually, it’s strange that the band from the first PCR didn’t show up again in this gel since I effectively just took the band out and put it back in if the PCR didn’t work. Increasing the elongation time helped clear up the smearing issue, but the yield band is still pretty faint in reaction 2 (first round). The same issue with the contaminated primers is present here in the controls.
I cut out the ~2200bp band and the ~200bp band from reaction 2. I cut out the bright band to test the gel purification kit. The slice with the ~2200bp band yielded 3.6ng/μL with a 260/280 of 3.29 and the slice with the ~200bp band yielded 23.7 ng/μL with a 260/280 of 1.92. Both were in 50μL of elution buffer and the curves looked the same as the one previously (peak at 230nm, flatline after that). It looks like something may be going wrong with the prep kit/my technique with the prep kit. If the gel preps aren’t working, that could lead to a failed round 2 PCR, though.
Fourth try:
This time I tested both Grace’s and Tony’s templates and I tried using the new primers that just amplify the Kan fragment without adding on the homologous ends. I tried not using BSA in these reactions to see if it would make a difference. Finally, I reduced the total number of primers used and added only 1μL of each primer (10mM) initially and added 1.5μL after 5 cycles to try to reduce the brightness of the primer bands.
Each reaction contained 30μL ddH20, 5μL 10X Taq buffer, 1μL 10mM dNTP mix, 2.5μL of each primer (10mM), 4μL 25mM MgCl2), and 0.5μL Taq Polymerase.
Reaction 1: 100ng of Kan plasmid from Grace Primers: 74mer UPTAG, 74mer DOWNTAG
Reaction 2: 100ng of Kan plasmid from Tony Primers: 74mer UPTAG, 74mer DOWNTAG
Reaction 3: 100ng of Kan plasmid from Grace Primers: KANMX-U2, KANMX-D2
Reaction 4: 100ng of Kan plasmid from Tony Primers: KANMX-U2, KANMX-D2
Reaction 5: 500ng Genomic DNA Primers: ACT1 SHORT (F & R)
Reaction 6: No DNA Primers: ACT1 SHORT (F & R)
PCR cycle profile:
- 94⁰C – 4 min
- 94⁰C – 1 min
- 55⁰C – 1 min
- 72⁰C – 3 min
35 cycles - 72⁰C – 20 min
Lane 1: Reaction 2
Lane 2: Reaction 3
Lane 3: Reaction 4
Lane 4: Reaction 5
Lane 5: Reaction 6
Skipped lanes were not counted in numbering.
Reaction 1 rolled off the parafilm as I was trying to load it. Sad day.
I don’t know why Reactions 3 and 4 have different length bands if they’re using the same primers. The Kan bands should be the same length even if the plasmids are different sizes. The reactions with Tony’s plasmid look about the same as previous first round reactions using Grace’s plasmid. It would have been nice to be able to compare this reaction with Grace’s plasmid to the one that jumped off the parafilm, though. I don’t know why there is such a definite band in Reaction 3 at ~300bp. I don’t think it’s primer bands because then they would be present in Reaction 4 as well because they used the same primers. It looks like BSA isn’t needed to get nice bands. I didn’t get a band in my negative control this time (I used ACT1 short primers instead of the HSF short ones), so it looks like the HSF1 short primers were contaminated. There should have been a more definitive band for the positive control reaction.
I cut out the ~2200bp band from Reaction 3 and purified it. The yield was 3.8 ng/μL in 30uL of elution buffer. The 260/280 was 1.57 and the curve was the same as the other ones (peak at 230nm then flatline). I don’t know why the concentrations have been so low. I’ve been careful to put the elution buffer right on the membrane. The only other thing I can think of is to try to elute with water instead of the elution buffer that is provided with the kit (Tris-Cl).
Day to Day Summary:
Tuesday 11/17 – 1.5 hours
Wednesday 11/18 – 0.5 hours
Thursday 11/19 – 3 hours
Friday 11/20 – 3 hours
Monday 11/23 – 6 hours
Tuesday 11/24 – 3.5 hours
Total Hours: 17.5
Open Questions:
I previously stated “… This probably means that the HSF1 short aliquots of primers are contaminated because the negative control using ACT1 short primers used in the previous series of PCRs worked well. The contamination could also be from using the BSA.” I would like to know where this contamination is coming from and if it can be eliminated.
Yields are low from the gel prep kit. I would like to know what the expected yield is from such a kit. 80% input DNA? 90%? 50%? I could run an experiment where I loaded some known amount of fragment DNA on a gel and purified (comparing before and after). Any other ideas or references?
Are there elution conditions or reagents that will improve the yield from the kit?
What is the purpose of BSA in these PCR reactions, anyhow?
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