I used a protocol to amplify a segment of a viral RNA. After making cDNA, I was able to detect the positive control four times with excellent bands, each time i used the same reagents including the Taq. In all, i have thawed the reagents more than seven times but did not place on ice (on the bench for at least 15mins). Now, i am not getting any amplification again even with the same cycling conditions, primers, nuclease free water and Taq. What is likely to be my problem?
I agree with Shouvik. You need to be very careful with your RNA and if using it multiple times you should aliquot the extract into multiple vials, thawing each a limited number of times to reduce degradation. Always keep your reagents and template on ice. Likewise with the reagents, it's good practice to aliquote those also, in case of contamination or, for example, if you forget to refridgerate them. At least in this way only some of your regents are compromised.
Check also that the tubes you are using for your PCR (and the tips) are RNAse free.
Good luck
Baner and Kelly, i must add that i used cDNA which is suppose to be more stable.
Hello Anyebe, quick question first, did you inactivate the RT after producing the RNA? All polymerases in the lack or absence of nucleotides activate their nuclease activity (which is needed for error correction during polymerization). Normally if you don't inactivate thoroughly the RT at 80 °C for 10 min your cDNA will be in pieces after a few cycles of thawing and freezing.
Concerning your PCR reagents, as Laura says, aliquoting primers and dNTPs is very good practice. In this way you can also work at room temperature, and you will limit the times you use the same reagents. dNPS as well as diluted primers (10-20 uM) are sensitive to thawing and freezing for chemical reasons (the phosphodiester bonds are easily subject to nucleophilic reaction with OH groups... and you always have free OH groups in water, even if not many at pH 7) and physical reasons (crystal formation during slow freezing of water can break the primers).
Finally, concerning the RNA, I can suggest a little trick. Keep it in Ethanol (so, the way you would precipitate it, with 0.2M NaOAc pH 4.5 and 2.5 vol. of EtOH), and just take out and spin down the amount you need for the experiment... it will always look as the first time you prepared it.
Dear Anyebe,
May be the reagents are the problem. Because you have prepared cDNA nicely, as your saying you got the bands in earlier reactions.
I will suggest to use a new set of chemicals for PCR. Taq and dNTP's are crucial. Always try to thaw it on ice. Put the reaction as fast as possible. Hope that you will get the result. and as suggested by Pietro and others, always use the aliquotes of chemicals, which save your efforts and lot of money as well.
Cheers!!
If I understood your question, you did not get a band with the same preparation of cDNA. Therefore, even if RNA is quite unstable, here it is DNA you handle . Even if stable it can be degraded also. If you take the cDNA from a unique sample and if for some reasons you contaminate the sample !!!! You should aliquot your cDNA and when it is no more working change all your products and also the cDNA.
And for safety it is better to keep even if DNA in ice after thawing and put it back to -20 °C rapidly. And also I wonder the length of the amplicon.
Throughout this week, i checked to confirm that the cDNA kit and RNA extraction kit are working. This I did with fresh RNA to test Bmicroglobulin. I also confirmed that the primers are good by running them on 2% agarose. The suspicion is that the antigen extracted to make the cDNA must have degraded due to frequent thawing and that other cDNA must have degraded due to temperature fluctuation. I went on to do a fresh extraction of sample taken from -56°C and reconstituted a fresh antigen for RNA made cDNA. Will run PCR next.
I hope it will work if you change & control everything and start from new cDNA preparation from a good RNA sample (that you have to check also). What do you mean by "a fresh antigen". Catherine.
Fresh antigen, meaning a new antigen.
Wow! I thank you all, respected colleagues and great researchers for your wonderful comments. After running the PCR using fresh antigen, I have now been able to reproduce the expected bands. I am so grateful to you for your useful suggestions which helped me to achieve this fit. I thank God I can still use my kits which can run up to 130 reactions.
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