Hello Everyone,
I am trying to amplifying a gene from the cDNA. I used Superscript III to prepare cDNA and got 1.5 ug/uL concentration. For amplification, I used 50 ng of cDNA template. But I did not get the band.
Cell line used to extract mRNA: 293T
My gene size is 5 kb
PCR recipe
cDNA template: 50ng
Proof-read enz: 0.5 uL
Buff: 1 uL
dNTPs: 0.5 uL
Forward primer: 1 uL (100 pmol)
Reverse primer: 1 uL (100 pmol)
Water: final vol to 10 uL
Total: 10 uL
PCR conditions:
Annealing temp: 60 deg, (Tm of forward primer is 70.1 deg and reverse is 61.1 deg).
Extension time: 5 min 10 sec, and
34 cycles.
Please shed some light on this.
Many thanks
Arun.
Does your gene contain a lot of GC ?
Which polymerase you are using ?
Try doing a gradient with different temperatures!
Dear Charbel,
GC content is 45% and I'm using EF taq polymerase (Solgent). I have tried gradient PCR with different temp ranging from 52 to 60 deg C but did not work.
you use a lot of primer. you should dilute your 100 p mol primers to 10 p mol and for reaction valume of 10 ul use only .5 ul of each primer
Test if your cDNA and other reaction conditions are good. Try to amplify an easier and shorter gene like actin from the same cDNA. This will serve as your positive control that your cDNA, DNA proof read polymerase and other reactions conditions are good.
If the above works then the next question is: Do you know that 293 cells express your gene? Maybe you want to try a cDNA from different cells that express your gene.
I think the problem is that you can't quantify cDNA because you are getting the concentration of the dNTPs and primers that are far more compared to your cDNA. So by using 50 ng you are not having exactly 50 ng of your template and the quantity you are using is not enough to amplify you gene of interest..
1. Search your gene expression level in 293t cell in some database(eg. The Human Protein Atlas. http://www.proteinatlas.org/ ).Optimize your PCR condition only when its mRNA level is not low, or you'd better change another cell line or tissue which express better to prepare the cDNA.
2. If your gene express high enough, the bottleneck may be the 5kb gene size. For this aspect, you can find a restriction enzyme site in the midle of the gene to seprete it into two 2~3kb parts. Clone them in two vectors sepretely, and then subclone them together in your final vector.
Good luck !
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