I have a PCR product of roughly 6kb (gene of interest + vector) that I cannot seem to grow colonies with in XL10 Gold cells using the Stratagene QuikChange II XL Site-Directed Mutagenesis kit..
I carried out a PCR scheme as follows:
5uL 10x Rxn Buffer (kit provided)
1uL (25ng) Template
2uL FOR primer (10uM)
2uL REV primer (10uM)
1uL dNTPs (10uM) (kit provided)
1uL PfuTurbo Polymerase (kit provided)
dH2O up to 50uL volume
Cycle:
95*C 5min
95*C 50sec; 60*C 50sec; 68*C 5min42sec (18x)
68*C 7min
4*C HOLD
After the PCR was complete I added 1uL DpnI (10 units/uL), gently mixed, quick spin, and incubated at 37*C water bath for 1hr.
Next, I thawed the XL10 Gold on ice (towards the very end of the 1hr incubation), added 2uL of B-ME and put on ice 10min, gently mixing every couple minutes. Then, I added 2uL of the DpnI/PCR product, incubated on ice 30min while mixing gently every few minutes, heat shocked at 42*C for 30sec, put back on ice for 2min, added 500uL of enriched SOC media, incubated at 37*C (shaking) for 1hr, growth observed. After the incubation I pelleted at 2000g for 2min and resuspended in 150uL of SOC.
Lastly, I plated 150uL of this resuspension onto LB plates (Amp) and put into the 37*C incubator overnight. I was expecting to see the plate blown away with that much being plated, but instead saw a plate that mirrored my neg control with no growth aside from a few tiny specs.
Any and all comments or suggestions are appreciated! Thanks.
Dear Corey,
The transformation efficiency of XL10 chemiocompetent cells is so high that I don't think that the this problem regards them. Eventually, you can transform an aliquote with a control plasmid for checking the transformation efficiency. Maybe, the problem is in your PCR reaction. Check if the amount of your primers is correct and if they were designed following the manual instructions. Moreover, try to increase the cycles of amplification up to 23 in order to increase the yield of the amplicon.
Good luck
Manuel
Hi Corey,
I never liked the Quickchange method for the very reasons you mentioned and abandoned it about 15 years ago. I use PCR to make 2 halves of the plasmid followed by homologous recombination. Its cheaper and always works. Moreover, you don't need to cleanup the PCR product or do any DpnI treatment to eliminate background. E. coli puts the 2 pieces together seamlessly. If you use a proofreading polymerase like Phusion or Q5 from NEB., and you just don't see any errors. I always use the 2x hot start master mix for the PCR steps.
You can use the method for mutations, insertions, deletions or even for combining two different plasmids to make chimeric genes. You can even put together 3 pieces of DNA as long as there are ~15-20 bp of overlap between joining ends. You can make your own competent cells and they will work just fine for well over a year for this type of work.(using the PEG-DMSO method).
The basic method for mutagenesis was first published as a high throughput technique. (BioTechniques 25:764-772 (November 1998).
Good Luck,
Stephen
Hi Corey,
I always run an agarose gel after mutagenesis, to make sure the reaction worked and I can see clear bends it the gel after DpnI treatment.
If you can't see bends (at a similar height as your template), or you see smears, this means the PCR reaction was not very successful. You might still get several colonies after transformation (and even with the right mutation), but what I usually do is try to change the annealing temperature, change the template:primers ratio (50ng:125ng usually) or number of cycles (20-30). If all fails - try changing your primers (at least one pair of G/Cs at both ends).
Hope this helps.
Good luck.
Hello,
I am using the Mutagenesis Kit you have mentioned right now and it works like a beauty each time.
I trust that your PCR conditions are correct. I follow slightly different protocol afterwards.
First of all I treat my PCR samples with the DpnI enzyme only 10 min and that show sufficient - I only get about 5% contamination with template DNA at worse case.
Second of all the B-ME I incubate only 2min on ice, then when I put my PCR reaction I mix the samples at the beginning and then leave it on ice for 30min and not disturb it any longer.
I also use LB medium rather than SOC.
After 1 hour incubation (after sheaking at 225rpm at 37) I never pellet my cell. Just plate out 100ul.
I am not sure if all of this makes any difference in the end but I get brilliant results each time.
I am surpriesd that you are not using controls - the kit comes with the plasmid control to check the efficiency of the bacterial transformation. If that works well then there is nothing wrong with your transformation procedure and your PCR has failed. Also you could use some other plasmid (preferably 6kb) to transform and if that works well then you also will know that the issue is your PCR and not the bacteria. I would advice to start there and rule out the problems.
Hope this helps a bit.
Hi Corey
I think your pcr parameters should be like this
95*C 30sec
95*C 30sec; 55*C 30sec; 68*C min ( 2min/kb ... in your case it is 12min) for 15 cycles
You should not thaw the competent cell for 1hr, instead you can thaw cell only 5 min on ice. prolong incubation may decrease the competency of the cell. When you incubate the cell after adding the pcr product, you need not to shake every few min. Just mix it one time and put it on the ice without any disturb. For 42 degree C heat shock, you can increase the time to 45 to 60 second.,
If you think it is the problem for competent cell, you can use DH5alfa or JM101
Best Luck
We recommend checking that the PCR amplification worked by agarose gel electrophoresis. If you see a clear band, yet no colonies: perform a quick PCR cleanup. We find PCR impurities oftentimes interfere with transformation efficiency. Make sure your transformation is working well by transforming a ready-made plasmid in parallel. Best of luck and happy mutagenesis!
Bruno Fonseca
http://primergenesis.com
Hi Corey,
I agree with the above comments that the problem is your PCR amplification, not the XL10 gold competent cells. I have been using the same Mutagenesis kit for the past one month to make different mutations. The first 2 run didn't work, I have to make changes to the PCR conditions (about 7.5kb full length plasmid ):
95*C 2min
95*C 50sec; 60*C 50sec; 68*C (70sec/kb) (18x)
68*C 7min
4*C HOLD
After I changed the PCR condition, everything works fine following the kit manual.
Good luck.
Ping
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