MEGAPRIMER PCR

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MEGAPRIMER PCR

Postby Andres_69 » Jan 31 2012 4:14 pm

Hi!

I´m trying to join two DNA genes in order to create a chimeric one. I´m using the megaprimer methods. The templates are one of 930 pb and the other 700pb. The have 15nt in common. I have try different Tannealing from 58-68 and i found a condition where i obtein a band in near the expected MW but i have really poor yield...I purified the product and try doing a 3rd PCR with the flanking primers, but i had no success. Do you have any advice? may be 15 nt is not enough and i have to add more nt in common, what do you think?

Thanks for your commets

Andres
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Re: MEGAPRIMER PCR

Postby mchlbrmn » Jan 31 2012 8:52 pm

The overlap must meet all the usual criteria for primer selection. The anneal temp as usual should be below the Tm. 15 nt is shorter than usual, but could work if the GC% is high, and the ends don't also anneal elsewhere.
Yes, I haven't seen the sequence, but my first guess would also be that the overlap may be insufficient. You could also try a lower anneal temp, with perhaps a bit longer anneal incubation. What is the Tm for the overlap?
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Re: MEGAPRIMER PCR

Postby Andres_69 » Feb 01 2012 9:07 am

This is the analysis of the overlapping region

SEQUENCE:
5'- AGC AGT CAC AAC ATC -3'
COMPLEMENT:
5'- GAT GTT GTG ACT GCT -3'
LENGTH: 15
GC CONTENT: 46.7 %
MELT TEMP: 45.3 ºC


The lowest Tanneling I have tried is 55°C, I was thinking of trying a lower Temp but I never did it, maybe it could be worth trying and incresing the time too.
I have tried different [ Megaprimer] and different [template] but I never obtain a band when I purified the product and did a 3rd PCR with the flanking primers.

Now, I order 2 more primers in order to increase the overlapping region to 75nt and the % GC to 50%..

I´ll try your advice of lowering the Tanneal and increasing time and tell you how it works.

Thanks for your helps!!!

Andres
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Re: MEGAPRIMER PCR

Postby mchlbrmn » Feb 01 2012 10:00 am

The calculators I used set at 1.5 mM Mg++, 0.2mM dNTPs, 0.25 uM primers give Tm estimates of 51.5C and 50.1 for this 15 nt sequence, so an anneal temp of maybe 47 would be called for, although because that is so low and I'd have to check the minimum temperature your enzyme can work at (you could ask the company), you could also try pushing it up a little more to 52C max, and perhaps extending the anneal time (if you wanted to play around instead of just waiting for the new primers). If your terminal end flanking primers are larger than this they run the risk of mispriming at this low anneal temp if closely matching sequence exists elsewhere in the template (I guess it's not a great risk because the template is not complex). It's usual to have an overlap of the same minimal parameters as the flanking primers they must work together with in the final PCR, usually around 20 nt and Tm of at least 55-60.
(I should have begun my first response by saying that I don't know the term "megaprimer", but I assume you mean fusing together two PCR products by PCR).
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Re: MEGAPRIMER PCR

Postby r.rosati » Feb 01 2012 12:20 pm

It's curious that you find the product, but just a few, and that you can't re-amplify it with a third PCR. The primers you used in the first-round PCR, to amplify the megaprimer.... you didn't switch tubes for the forward and reverse primer, did you? If you had to amplify the 2nd round with the forward and you're adding the reverse, or vice-versa, then only the megaprimer DNA will function as rightful primer (hence the few product at the right size) and a third PCR won't work, as per your findings.
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Re: MEGAPRIMER PCR

Postby Andres_69 » Feb 01 2012 3:31 pm

They say megaprimer pcr, because one amplimer serves as a huge primer but with the double strain difference of a common primer.

I tried different approaches

A)30 cicles with only template and megaprimer.
B)Adding only the reverse primer in the first PCR in the annealing step after 5 cicles and continued 30 cicles
C)Adding both flanking primers in the first PCR in the annealing step after 5 cicles and continued 30 cicles

B and C is what they call Megaprimer Method.

when I use both flanking primers at 57 and 64 C° of Tanneal, I obtain a broad band near the expected size, as you can see in the picture
Image

Then when I purified and try another pcr I do not have any kind of amplification.

Now I testing the changes in the annealing step, I use

Time I extend from 2 to 3 min
Tannealing from 44 to 52 °C

Tomorrow I´ll run the gel and tell you how it works..

Thank you so much for your help!

Andres
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