Methods Digest, Vol 56, Issue 10 (Message 2:Joining two PCR products together)

Liyana Ismail via methods%40net.bio.net (by liyana_ismailmr from yahoo.com)
Mon Jan 11 15:53:14 EST 2010


Hi Catherine,

I'm tempted to reply to this one since we're doing something pretty similar.

I used High Fidelity PCR (Fermentas) to generate two PCR products from cDNA cloned into pGEM-T vector, 5'-end region (1000bp) and 3'-end region (400bp). 5'-end region and 3'-end region has overlapping region of 319bp.

After PCR product purification (this is important step to reduce smears significantly), I had used forward primer of 5'-end region (SP6) and reverse primer of 3'-end region (oligo (dt)17 adaptor) to generate single PCR product, sized 1200bp when viewed in 1.2% AGE. I'd found out that in my case, too much cycles will produce a lot of smears.

After optimization, I had managed to get the desired single PCR product by doing touchdown annealling for 10 cycles with all the PCR components except primers. This is followed immediately with addition of flanking primers by 25 cycles similar PCR reaction profile I used to generate 3'-end region (this have higher annealing temperature) with addition of 30 seconds to denaturation and anneling steps, and 1 minute for extension step. 

I only used 0.5ul of each templates and 1ul of each primers (10uM/ul). I didn't have the concentration of the initial PCR product, but after purification step, the bands is much brighter then the DNA ladder*. Total volume of PCR mixture is 50ul.

*I used 5ul DNA ladder (0.1ug/ul).

I hope this could help you.

Cheers,
Liyana

Genetic Engineering Lab,
Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak,
94300, Kota Samarahan,
Sarawak, Malaysia.




________________________________
From: "methods-request from oat.bio.indiana.edu" <methods-request from oat.bio.indiana.edu>
To: methods from magpie.bio.indiana.edu
Sent: Tuesday, January 12, 2010 1:04:47
Subject: Methods Digest, Vol 56, Issue 10

Message: 2
Date: Mon, 11 Jan 2010 15:32:31 +1100
From: klau from med.usyd.edu.au
Subject: Joining two PCR products together
To: methods from magpie.bio.indiana.edu
Message-ID: <20100111153231.188828c1ms8dodz4 from www.mail.med.usyd.edu.au>
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Dear Dr. Giles,

I came across your answer regarding how to join 2 pieces of PCR  
products together in a single cycle PCR. Please advice me if this is  
the right way to produce a single piece of DNA fragment from two PCR  
products. Here is my experiment:

1. From cDNA, I have generated two PCR products using two different  
primer sets. One product has a size of 500bp (A), while another one is  
600bp (B). A and B has around 10-15 homologous bases.

2. Consequently, I need to generate single PCR product using the  
forward primer of A and reverse primer of B, to produce a final  
product of approximately 1100bp. I have repeated this stage for many  
times but still failed. What I get is just smear. I only performed  
normal PCR without the 2 quasi-exponential PCR as what you have  
mentioned before.

3. Do I need to perform 2 quasi-exponential PCR for the initial PCR;  
to produce A and B? Can you please explain the PCR parameters in  
detail? Can I replace it with a normal PCR, i.e. initial denaturation  
(98C, 2min), followed by 35 cycles of (98, 30sec-> 55C, 30sec-> 72C,  
30sec) and finally final extension at 72C, 8min.

4. With the initial PCR product, do you recommend me to use it at a  
small quantity (how much would be sufficient?) to perform a single  
cycle PCR without adding the primers at high Tm? Does that only  
include these steps: 98C, 30sec-> 70C, 30sec and 72C, 30sec, for one  
cycle? Do I need to add DNA polymerase at this step?

5. Do I then use this single cycle PCR product from above as a  
template for exponential PCR or just add in appropriate primers and  
run 15-cycles exponential PCR for this product at primers Tm? Can you  
explain the exponential PCR here in details?

Thank you so much for your help!!

Cheers,
Katherine

Kolling Institute of Medical Research
Royal North Shore Hospital
St Leonards, NSW, Australia, 2065



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