Re-PCR trouble

[Marina Ntika]

Hello everyone.

I am trying to amplify a gene, and at the same time to introduce point mutations, to enable the digestion with a specific enzyme of interest, with the ultimate goal to clone my gene into a specific plasmid.

Since the point mutations I need are 5 (couldn't find a more appropriate site to design primers, cause I need to stay in frame with the target-plasmid's ATG) I designed two forward primers, the first with 3 point mutations and the second matching the first forward, and introducing the remaining two mutations. I use the same reverse primer to pair both my forwards.

The first pair (forward primer A and reverse primer) worked just fine, I got the product I wanted, easily and quite clear (after fine-tuning the profile I got only one band, the right one). However, during the re-PCR using the previous resulting product as template, and using the second pair of primers (forward primer B and reverse primer), I get 2 bands, both of them extremely intense and both of them quite lower than the expected one. With higher concentration of MgCl2, I got also a third band (the desired one) but it is extremely faint. I cannot be sure if it is also some portion of the template DNA that didn't react with the primers...

The amount template for the rePCR is 1ul.

The annealing temperature for both pair of primers is 64C, but I have gone up to 66, and still, first pair works, second doesn't.

Also, a quick note: Introducing the mutations by alternate ways or selecting a different enzyme for the cloning procedure is not an option.

Please, any clues and suggestions are mostly welcome.

Sorry for the long post, and thanx in advance.

[kmunson779]

Am I reading correctly that you used 1 uL of your first PCR product as a template for your second reaction? This seems like an awful lot of DNA to me.

How did you purify your DNA between the two PCR steps? How much DNA (in terms of moles or grams) are you adding to your second PCR reaction?

How many cycles are you running for the two reactions?

I know you say you can't use any alternate methods, but it may be worth blunt-end cloning the first product to verify it has the correct mutations before proceeding.

It also may be worth designing another reverse primer outside the region you want to amplify for the first PCR reaction, to avoid problems you may have trying to re-prime with the same primer.

[oBWhat]

Yes usually we dilute the 1uL of the PCR reaction in 10ul of purified water and then use 1ul of that for another PCR, as the PCR mix can inhibit your reaction as well.

[Marina Ntika]

Originally Posted by kmunson779

Am I reading correctly that you used 1 uL of your first PCR product as a template for your second reaction? This seems like an awful lot of DNA to me.

How did you purify your DNA between the two PCR steps? How much DNA (in terms of moles or grams) are you adding to your second PCR reaction?

How many cycles are you running for the two reactions?

I know you say you can't use any alternate methods, but it may be worth blunt-end cloning the first product to verify it has the correct mutations before proceeding.

It also may be worth designing another reverse primer outside the region you want to amplify for the first PCR reaction, to avoid problems you may have trying to re-prime with the same primer.

Well, I didn't actually purify the first PCR product before proceeding to the second one, cause my band was very clear, and I had no other products, or smear. However, I plan to try again and extract the template for the second PCR through gel extraction, just in case a non-visible band is being amplified as well. The template I use for the second PCR is about 10-30 ng. I run 40 cycles.

Unfortunately, I do not have time to redesign and order primers, cause I have to present my diploma work on September, and I still have a second cloning part (with the FLAG gene) and then the whole gene (FLAG-initial gene) must be cloned into a given expression vector, do a large scale isolation and then go in vivo. My schedule is extremely tight!!! This lack of time, in addition to poor funding of the project leads to my inability to pursue alternative methods. Imagine that I don't have even the ability to use kits, I do all my isolations the old fashioned way, which is extremely time consuming.

Anyway, thank you very much for your reply, I will also try to dilute my first product 1:10 and try using this as template as well, as oBWhat suggests (thank you too), and see how it goes...