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: "[email protected]" <[email protected]> To: [email protected] 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: [email protected] Subject: Joining two PCR products together To: [email protected] Message-ID: <[email protected]> Content-Type: text/plain; charset=ISO-8859-1; DelSp="Yes"; format="flowed" 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 Get your preferred Email name! Now you can @ymail.com and @rocketmail.com. http://mail.promotions.yahoo.com/newdomains/aa/ _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
