I had done PCR with cDNA and got some non specific band. Now i have very limited cDNA, which is not enough to perform all PCR, So can i use my previous PCR product as DNA.
I have attached my Gel pic stained with EtBr.
If you have any other suggestion to resolve it then please write me.
With regards.
Thank you
yes you can re amplify but you have a large amount of product so even if you purify a band and re amplify it then only use a 1:100 dilution of the product and set up a few tubes and run 15-25 cycles only as each 10 cycles will produce about 1000 times more product and too much amplification leads to long smears of product annealing to product. If you know the sequence of your product try nested primers where the second round primers end about 10bases inside your first round primers which can be done on unpurified first round diluted product and will clean up the final amplimer
Dear Pravin,
If you have a more specific analyze after cDNA synthesis, you can use this PCR product as a DNA sample. But there are so many unspecific amplicon which cause unspecific reaction in second amplification. You should optimize your PCR reaction (annealing temperature, DNA concentration, purity or other factors) firstly. Also you can't know if your PCR work successfully with this image. So first of all you should optimize your PCR reaction and product a only one specific amplicon.
Hi.
You mean that you want to separate nonspecific bands from gel an reuse them?
It means that you want to do other pcr reaction with new primers.if I'm not mistaken .
Hello Niloufar Mohammadkhani
Thank you for your kind response. Your attached PDF file helps me in getting the facts.
yes you can re amplify but you have a large amount of product so even if you purify a band and re amplify it then only use a 1:100 dilution of the product and set up a few tubes and run 15-25 cycles only as each 10 cycles will produce about 1000 times more product and too much amplification leads to long smears of product annealing to product. If you know the sequence of your product try nested primers where the second round primers end about 10bases inside your first round primers which can be done on unpurified first round diluted product and will clean up the final amplimer
Amplification efficiency At the experimental level, the amount of product formed depends on of a prime factor that is the amplification efficiency (E) defined as the average proportion of target DNA molecules duplicating each cycle amplification. Amplification efficiency is included between 0 (no amplification has occurred) and 1 (after each PCR cycle, each target DNA molecule generated two amplicons). Be careful because exaggerated amplification will give you very long tapes. You can reamplify using a 1 in 100 dilution with a cycle of 15 to 25 is accepable.
- Badges
- Science method