Doing SDM, getting PCR but not getting transformation colonies. What could be the issue? Comp cells are fine when used control.
-PCR: 10 cycles (40ng plasmid template), Can See right size PCR band on the gel.
-PCR digestion with DPN1 (1ul 2hr)
-Heat Inactivation 72℃ for 20 min.
-For transformation 0.5, 1, 5, 10 ul of above mixture in to 30ul of JM109 competent cells Kan+plates. (Control plasmid total 10ng in 1ul volume for 30 ul cells(same) gives colonies)
So no colonies on SDM mixture. What could be the issue?
Dear Karan, most probably there is a problem in your PCR. At least this is what I've encountered many times. The most important are your primers parameters, mainly their Tm. I control these parameters using calculations specified in https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.agilent.com/cs/library/usermanuals/Public/200518.pdf&ved=2ahUKEwiC7dGq2p_4AhU_h_0HHVkeBI8QFnoECCAQAQ&usg=AOvVaw3Huwda5nQXJKt05DSrso9c, page 9.
not easy to answer your question because site directed mutagenesis is very powerfull; I would check first some very basic points (sorry)
-is kanamycin the right antibiotic?
-did you really use dpnI ? (and not another enzyme ...)
- the primers seems Ok as you have a PCR product; I would also control the PCR product after dpn1 digestion (if you see it before dpn1 you should see it after...but sometime you do not see anything and you have the right clones anyway..)
- but is it the right PCR product? is it possible that one primer hybridize somewhere else in the plasmid ? then you get a linear DNA (that can be favoured by the PCR because it is shorter) and not the circular plasmid DNA ...are your primers totally complementary (one direct one reverse: I usually design them to have at least 12 nucleotides after the last mutation and 12 before the first mutation I wanted to introduce.
you may increase your PCR hybridization T° by 5°C...
for site directed mutagenesis the number of PCR cycle is limited to 12-16 cycles first to avoid the introduction of unwanted mutations by the polymerase; second tbecause of the efficiency of the competent bacteria (10e8/ug= 10e5 clone for 1ng of DNA... so even if you don't see the PCR product you can get plenty of colonies in practice sometimes you see the PCR product sometime not but even if you don't see it you transform and you will be able to have mutants.
This could be primer binding where primes are binding somewhere else but still giving me right size band. There is no way to find this out. Yes I see right size band so assume that primers are giving full length PCR products but not sure if these are binding at the right place (Anyway to find out?)
Though, I did increase Tm for annealing from 59 to 62 and still getting band size. My primers are designed by https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools?gclid=EAIaIQobChMIjLTW9bWj-AIVRfLjBx1-tAHNEAAYASABEgJp2_D_BwE
I may try 63 and see if it will make more specific binding then.
So I assume the software tool would have surely consider length, overlapping region and Tm optimum factor while getting me primers pair.
So is there anyway I can check if my primers are binding at right location or not. I don't think there is anyway to do so!
10 cycles are enough because I do get band and when I compare plasmid template 1ul on gel (out of 50ul with 40ng in it), I don't see plasmid band so whatever bright good enough band I see is from PCR only.
Yes, I am adding DpnI only. Yes I confirm Kan plates with + and - stains to make sure plates are right and working fine.
Yes After DpnI digestion I don’t see any plasmid. As such I don’t see plasmid even without digestion because it is way too less (40ng in 50ul reaction).
I don't understand you wrote "PCR: 10 cycles (40ng plasmid template), Can See right size PCR band on the gel." and then "After DpnI digestion I don’t see any plasmid. As such I don’t see plasmid even without digestion because it is way too less (40ng in 50ul reaction)."... you see or you don't see??
I don't understand you wrote
"PCR: 10 cycles (40ng plasmid template), Can See right size PCR band on the gel."
Ans: I can see right size PCR which I intend to amplify.
and then "After DpnI digestion I don’t see any plasmid. As such I don’t see plasmid even without digestion because it is way too less (40ng in 50ul reaction)."... you see or you don't see??
Ans: This is the template plasmid not the PCR product. So PCR product of course I see but template DNA I don't see on the gel either Pre digestion or post digestion.
I hope I cleared your confusion. I think you got confused by not reading it clearly that I am writing PCR for the product outcome which is still PCR product unless get ligated inside the cells and make plasmid.
The Plasmid I mentioned here is the Plasmid template (from which PCR is getting amplified). This is not PCR because that is circular template Plasmid.
Let me know if I cleared this confusion.
- Badges
- Science method