Hello everyone,
We are currently doing some deletions in one of our plasmids and after obtaining colonies, we are trying to do colony PCR, but it gives us just some smear (incl. positive control). Considering that the marker looks good, it's likely problem with the PCR, rather than the electrophoresis.
At first, colleague did the PCR, her gel is on the picture named original. She got at least some resemblence of bands. Then I did new colony PCR and it's even worse.
My setup of the PCR was as follows:
5x OneTaq Standard Reaction Buffer 4 ul
10 mM dNTPs 0.4 ul
10 uM fw primer 0.4 ul
10 uM rev primer 0.4 ul
boiled bacteria 2.5 ul
OneTaq DNA Pol 0.4 ul
water 11.9 ul
This polymerase was brand new, we got it few weeks back, nobody used it yet. I used higher concentration of the polymerase, as they recommend for longer amplicons.
I did touch-down PCR, because the primers are not tested yet. The cycling was as follows:
94°C 30 s
94°C 20 s
54-0.5°C 30 s
68°C 10 min 10 x
94°C 20 s
49°C 30 s
68°C 10 min 30x
68°C 5 min
The elongation is so long, because I used the original plasmid as positive control, which should give amplicon of 7 kb with one pair of the primers. The other combinations of primers and templates should give amplicons between 1 kb and 4.5 kb.
I did two sets of primers, one is on the top row of the picture, the other is at the lower row of the picture. The positive control (which is isolated plasmid, not bacteria with the plasmid) is in the last well on top row and first well on lower row.
Any idea what could have gone wrong? Or should I try to change?
My plan at this moment is to try just with the positive control plasmid with my Taq polymerase, which I know works to check the primers. Otherwise I don't know.
Ideally the primers should anneal much hotter as the plasmid will be coiled and supercoiled making pcr difficult unless the plasmid were cut and opened up between the primers so maybe design primers either side of a unique cut site and restriction digest before pcr. If this is not possible then run the pcr under denaturing conditions such as 6-8% dmso or 1molar betaine in the pcr mix. Run the gel slower for better visualisation of the results. Ideally use longer primers but at least run a temperature gradient to determine the highest temperature that will give you a pcr product to help reduce the secondary structure of the plasmid
Hi,
I'm not sure I follow. Do you think that my annealing temperature was too low? It was setup based on NEB's Tm calculator.
Also, the bacteria wee heated to 98°C for 10 mins, so that shouldn't be supercoiled, should it?
Should I use the GC Reaction Buffer and/or High GC Enhancer?
I have no problem with a low annealing temperature when amplifying simple, short linear dna but plasmid is coiled and supercoiled ( when the temperature drops from 98 the coiling is created as the temperature falls) and the coiling is less at higher temperatures which is why I prefer longer primers so that the annealing temperature is kept high at all steps of the pcr minimising secondary structure formation. Either of GC enhancer or gc reaction buffer will contain one or both of dmso and betaine so either will help in the pcr of complex folded dna samples
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