PCR to sequencing problems - (Jun/26/2008 )

Hi,
Im having problem with isolating and sequencing a specific gene. The basic outline is as follows:
1. RNA isolation from tissue
2. RT-PCR to create cDNA copy
3. Purify the product using Qiagen kits
4. TA cloning
5. Insertion of gene into plasmid pIRES puro
6. Use Blue white screening to select cells with genes
7. Isolate the DNA to be sequenced.

Now, im having a few issues with the purification. I have optimized the RT PCR by playing around with the RT temperature and also the annealing temperature to get better yield, however im not getting very pure product after the PCR coz when i run it on the gel, all i get is a band with a tail behind it if you know what i mean.

The primers we are using have been specifically designed to target the gene of interest but after receiving the sequenced data, the wrong gene was amplified. Any comments about how to get the gene i need amplified and sequenced or any other trouble shooting hints and tips for that matter. All comments welcome!!

Thanks!!

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Hello!!
Try to purify the product two times using qiagen kits or improve your pcr..

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Hey, I tried that, well i purified the product first and didnt get a very clean band so ran a gel purification and lost all my product in the process.

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Try a different RTase. For some reason, some transcripts work better with one enzyme compared to another. BTW, which enzyme are you using?

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Im currently using Taq.

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hum... as a polymerase or as a RTase ?

anyway... I don't know what you mean by "a band with a tail behind"... could you post a picture of you gel ?

It might be that you are loading to much DNA on the gel...

plus, I guess it could be useful to provides informations about your template... it's then easier to think of what could have contaminated it (if not your human DNA ... )

and did you send more than a single clone to sequencing ?

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