Biochemistry question about PCR amplification and cloning of P.profundum alphaCA
ID: 212556 • Letter: B
Question
Biochemistry question about PCR amplification and cloning of P.profundum alphaCA and beta CA. No DNA was obtained in the gel electrophoresis that was ran after the PCR. Please help me troubleshoot what went wrong, and tell me how I can check if that was what went wrong. Procedure for PCR: (all vol microliters)
1. 25 PCR Master Mix(from a kit), 1 forward primer(CAalphaP1), 1 reverse primer(CAalphaP2), 10 microliters P.profundum DNA template, 13 nuclease free water; all combined and vortexed, centrifuged, vortexed, centrifuged, then put on ice for a few minutes.
2. Put in thermocycler, 98C for 30 sec, then (56C for 30 sec, 72C for 30 sec, 98C for 10 seconds) repeat 30 times, then 72C for 5 minutes, then held at 4C for a week.
3. For gel 1.5% gel was used, from agarose and TAE buffer. 4 microliters flourescent dye added to 20 microliters DNA mixture from PCR parafilm method. Placed into wells. Ran 30 min at 110V.
At this point I did not see any flourescent bands under the UV light, making me wonder where my DNA went. HELP!!
Explanation / Answer
All of the settings mentioned above seems to be correct and you should be able to get the amplified band. Its just that i am not sure about the fluorescent dye that you used to observe the DNA.
We use EtBr dye to visualize DNA on gel and we do not add the dye directly to the sample, we add the dye to the gel and allow the DNA to take the dye as it is running in the gel. This might be the issue, or you can tell me the type of dye you use, because i havent used any other method such as yours.
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