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Polymerase for E. coli screenings

polymerase

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5 replies to this topic

#1 pDNA

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Posted 27 July 2012 - 03:44 AM

Dear people,

can somebody recommend a good proof-reading enzyme that can be used for the amplification from genomic DNA (E. coli). We have done several modifications and we want to confirm them.
We have tried two Taq polymerase(HybriPol, a rather fast enzyme and recombinant Taq from Fermentas), Phusion polymerase and KOD. Phusion, KOD and recombinant Taq give use most of the time no product at all. The fast Hybripol gives us product but a lot of unspecific bands as well ...therefore not suitable for sequencing.

Maybe someone of you can suggest a good enzyme that has a good performance when using gDNA as a template.
I'm also open for general suggestions when using genomic DNA as a template. I'm really loosing nervs at the moment and any advice is welcome!

Many thanks in advance!

Best regards,
p

#2 phage434

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Posted 27 July 2012 - 06:28 AM

In general this has much less to do with the enzyme and much more to do with the primers and the conditions. How is your template prepared? How are your primers designed?

#3 pDNA

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Posted 27 July 2012 - 07:28 AM

primers are 18-23 bp long, i did already gradient PCR (with annealing temperatur from 50-70°C) ...not even amplification of the positiv control.
I try to confirm a knock out event ...so one primer binds upstream of the integration site and the other one is specific to the antibiotic resistance gene i use for the knock-out.
My template is prepared by lysing the cells using SDS and protease K, incubate at 37°C for one hour and then purify the lysate using ZYMO DNA clean & concentrator spin columns, DNA is eluted in water. The template is high molcular weight DNA (checked on a gel) with concentration varying between 100-300 ng/µL. Already used different template concentrations (100-500 ng).

Many thanks for your time and help!

Regards,
p

#4 phage434

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Posted 27 July 2012 - 04:20 PM

I'd say that is conclusive evidence that your primers are either incorrectly designed or bad. You should be able to screen E. coli with much less effort. Typically I pick a colony with a pipet tip into 50 ul of water, mix, and then use 1 ul of that in a colony PCR reaction. Extend the initial denaturation period to 5 minutes to lyse the cells, then do a normal PCR. There can be some false negatives, but few false positives. You may be using far too much template genomic DNA in your reaction. Try doing a 10x 100x and 1000x dilution series on your DNA. Too much DNA is a strong inhibitor of PCR.

#5 Ameya P

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Posted 28 July 2012 - 12:13 AM

Hi pDNA,

Like Phage said, it looks more likely that your primers are bad/ not working than the fault of the polymerase. Diluting your DNA further should also help.

In case all has failed, I have used Sigma and Bioline Taq (Cat 21041) on gDNA sample and obtained good results from sequencing too.

Hope your reaction works out soon.

Ameya

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#6 pDNA

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Posted 28 July 2012 - 01:22 AM

many thanks for your suggestions so far!

I can rule out that the primers arn't working (i'm an experienced molecular biologist i would say and different primer sets have failed). I have also tried different dilutions of the template (very low and high concentration) without success.

Is it possible that the gDNA purification (using crude lysate on spin columns, lysed with SDS and proteinase) is the problem? ...i already performed many PCRs (gap filling project for an E. coli genome) without serious problems ...the only difference was that the template was purified using the CTAB method and phenol/chloroform extraction followed by isopropanol precipitation.

Is it possible that i have SDS carry over that ruins my PCR reaction? Are there enzymes available that are more insensitive to inhibitors (like the hemo klen taq from NEB) that have been used by some of you?

Many thanks again!

Best regards,
p





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