4 replies to this topic

[Fiona P]

This is incredibly frustrating for me. I tried unsuccessfully several times to amplify a PCR product using overlap extension and the following reaction mixture:

10, 20, 50 or 100 ng template DNA (you can see it on a gel)
1.25 uL of 20 uM primer 1 (Tm ~65-69)
1.25 uL of 20 uM primer 2 (T7 term or T7 pro, both of which are sequence-verified on pSGX3 plasmid)
1 uL 20 mM dNTPs (made in 100 uL aliquots)
10 uL of 5x HF buffer (also tried GC buffer, also tried other PCR buffers and then added Mg++)
0, 1, 1.5, 3, or 5% DMSO
0, 0.5, or 1 uL of 50 mM MgCl2
1 unit Phusion polymerase or 1 unit Platinum Taq
50 uL total reaction volume using nuclease-free water
put into a thin-walled PCR tube (often used for gradient PCR) which has been autoclaved.

my PCR programs are pretty much from the Phusion or Taq handbooks with the only variation being the Tm. I've tried gradient PCR from 45 C to 70 C for the Tms, and nothing ever amplified. EVER. I used FailSafe PCR kits, I tried getting fresh everything... nothing ever changed the outcome.

So then I went to QuikChange (I did NOT buy the kit-- I found that Phusion had been used by other people and for a while they made Phusion QuikChange-like kits) and used pretty much the same mixture except using 20-40 ng DNA and always 1.5% DMSO.

After the QuikChange, I add 1 uL of DpnI and digest for about an hour (sometimes overnight) and then N-butanol ppt the DNA, electroporate into XL1Blues, and then plate. I get colonies on the mutant plates and no colonies when I DpnI digest the template. I got 5 colonies the first try, but come time for sequencing there's no DNA coming from my minipreps... there will be about 50 ng/uL (from NanoDrop), but if I wait a while and run a gel you can't see anything... best case scenario, you see maybe a band and maybe a blur. My template is perfect when I digest it, every time.

For the minipreps, I use 5-10 mL of LB with 5-10 uL of 50 mg/mL Kanamycin overnight (~16-18 hours) at 37C and 220 rpm. Then I miniprep using Qiagen kit (freshly purchased... threw away all my old stuff) and I always wash with PB. Then I elute with EB, and also tried eluting with nuclease-free water. Nothing is working.

What is going wrong with this? First the SOE didn't work, now the minipreps aren't working. I can't think of anything else to try... any suggestions? PCR is so mean!

[phage434]

I'd start by diluting your template 100x. Too much template inhibits PCR reactions.
Did you remember to add ethanol to the PE wash buffer on your minipreps?
Can yo do a control miniprep of a strain you know has a plasmid?
Might your strain by Kan resistant?

[qzlabs]

Fiona P, on 12 July 2011 - 02:29 PM, said:

This is incredibly frustrating for me. I tried unsuccessfully several times to amplify a PCR product using overlap extension and the following reaction mixture:

10, 20, 50 or 100 ng template DNA (you can see it on a gel)
1.25 uL of 20 uM primer 1 (Tm ~65-69)
1.25 uL of 20 uM primer 2 (T7 term or T7 pro, both of which are sequence-verified on pSGX3 plasmid)
1 uL 20 mM dNTPs (made in 100 uL aliquots)
10 uL of 5x HF buffer (also tried GC buffer, also tried other PCR buffers and then added Mg++)
0, 1, 1.5, 3, or 5% DMSO
0, 0.5, or 1 uL of 50 mM MgCl2
1 unit Phusion polymerase or 1 unit Platinum Taq
50 uL total reaction volume using nuclease-free water
put into a thin-walled PCR tube (often used for gradient PCR) which has been autoclaved.

my PCR programs are pretty much from the Phusion or Taq handbooks with the only variation being the Tm. I've tried gradient PCR from 45 C to 70 C for the Tms, and nothing ever amplified. EVER. I used FailSafe PCR kits, I tried getting fresh everything... nothing ever changed the outcome.

So then I went to QuikChange (I did NOT buy the kit-- I found that Phusion had been used by other people and for a while they made Phusion QuikChange-like kits) and used pretty much the same mixture except using 20-40 ng DNA and always 1.5% DMSO.

After the QuikChange, I add 1 uL of DpnI and digest for about an hour (sometimes overnight) and then N-butanol ppt the DNA, electroporate into XL1Blues, and then plate. I get colonies on the mutant plates and no colonies when I DpnI digest the template. I got 5 colonies the first try, but come time for sequencing there's no DNA coming from my minipreps... there will be about 50 ng/uL (from NanoDrop), but if I wait a while and run a gel you can't see anything... best case scenario, you see maybe a band and maybe a blur. My template is perfect when I digest it, every time.

For the minipreps, I use 5-10 mL of LB with 5-10 uL of 50 mg/mL Kanamycin overnight (~16-18 hours) at 37C and 220 rpm. Then I miniprep using Qiagen kit (freshly purchased... threw away all my old stuff) and I always wash with PB. Then I elute with EB, and also tried eluting with nuclease-free water. Nothing is working.

What is going wrong with this? First the SOE didn't work, now the minipreps aren't working. I can't think of anything else to try... any suggestions? PCR is so mean!

The concnentration of the template could be an issue. Also the extension time is very different between a taq and a phusion. Anealing temp for taq, tm+3, for phusion, tm-3. There should be nothing wrong with your miniprep except you might want to check the pH of all three buffers in the kit.

[sciencelover]

It may be your miniprep kit.. Try this Zyppy miniprep kit. Its a pellet-free procedure that bypasses conventional cell pelleting and resuspension steps. It produces much better yields than the Qiagen kit. It also has colored buffers so you can visualize bacterial cell lysis and buffer neutralization steps.

[klinmed]

Is your Mg++ conc. a little low? I would try 2 mM and 4 mM. This could be especially important if you have any EDTA in your template