getting long pcr from cDNA - (Jun/02/2008 )

Hi all,

I am new in working with mammalian tissues and cell lines. I have been trying to extract Total RNA from cell lines or brain tissue, using it for reverse transcriptase reaction to get cDNA for pcr of my gene of interest of about 2.7kb. I have either fail to get any pcr product or pcr product with very little yield. Can anyone advise how to pcr my gene of interest with higher yield?

Thanks

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1. Use the cells/tissues that have the maximum expression of your gene.
2. You may want to order RNA from a company.
3. Give about 3 minutes for PCR extension time.
4. Use RT that allows upto 55 or 65'C, in case if your RNA has secondary structure/s.
5. Order some more pairs of primers.
6. You can divide the PCR product in two, with a good cloning strategy.
7. If you are doing this just to get your gene, your full cDNA clone may already be avialable (check addgene). Just order a clone.
..

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Several things you can do. Cell counter has made some good suggestions there. Just to elaborate on some of his/hers:

1. Look up the paper/s that originally characterised your gene. These papers usually have a Northern blot or similar results that show the expression of your gene in a variety of tissues. This is the best way to get the expression levels of your gene in each tissue and choose the tissue with the most expression. Higher expression = more likely to amplify gene. If 1 uL of template doesn't work, use 2 uL or 4 uL of template, etc, to increase the number of targets of your gene. If the brain is the highest level of expression, try more specific areas of the tissue, in the case of brain - hippocampus, pituitary, hypothalamus, etc. Some of these areas will have even higher expression, brain is just an average of all its parts after all.
2. RNA or cDNA from a company is a good way of getting reliable template, it reduces a potential area where you could be going wrong and is good for specific tissues such as pituitary, etc where you can't make much yourself.
3. Extension time, GC richness is also important. If the template is GC rich use some betaine. Don't be afraid to use more cycles or reamplify your little amount of target if you can see it on the gel.
6. Look up splice overlap PCR. Two smaller targets are often more easy to amplify than one large target, particularly with cDNA because not all cDNAs may be full cDNAs, some will be partial length especially if using random hexamers to make the cDNA instead of poly-T primers.
7. Addgene, Open Biosystems (generally cheap), Origene (generally expensive). See how much they are, can be worthwhile just getting a clone containing the desried sequence and use this as the template although usually too expensive. This is a good backup/final resort. But i have faith you will get your gene.

Good luck, Rob

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Next to the given suggestions:

What RT are you using, what are your PCR-conditions, which polymerase are you using? Are your primers described before or how did you choose them?

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step cloning is a good idea. PCR out 500 bp at a time and splice them together.

as for PCR from cDNA, you need good quality RNA and cDNA. You need to use a Taq with proof reading. Error prone Taq will stall the polymerase and terminates the PCR prematurely.

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