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PCR help needed


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3 replies to this topic

#1 pilic8

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Posted 24 August 2009 - 01:20 PM

I have been working on troubleshooting my PCR reaction for a while. I am attempting to isolate a ~600bp section and have gotten no bands at 54 degrees of annealing, and non-specific bands at 50, 52 and 53.

I have gotten the primers to work on a closely-related species for the same fragment but can not figure out whats going on. Im working with mitochondrial DNA and the absorbance numbers are pretty good so I dont think it is the template. I attempted a few different concentrations of DNA at 54 degrees and they did not work.

Can somebody give me any suggestions that may push me towards the right direction? Im pretty new to the wonderful world of molecular technology so Im a bit confused on what to try next.

Thank you. ;)

#2 lab rat

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Posted 24 August 2009 - 01:53 PM

hi Pilic,

I'm guessing that you're isolating DNA from bacteria? Try adding adding a little DMSO to your reaction--here are the conditions I used for a 16S rDNA product:

H2O: 28.45 ul
Buff: 5.0 ul
MgCl: 4.0 ul
DMSO: 2.5 ul
Primers: 1.25 ul ea
dNTP: 1.0 ul
Taq: 0.3 ul
gDNA: 6.25 ul
____________
50.0 ul

regards,
lab rat

Edited by lab rat, 24 August 2009 - 01:54 PM.

42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#3 pilic8

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Posted 24 August 2009 - 02:17 PM

Thank you for the prompt response! Im actually isolating from an echinoderm, and Im using a Taq kit from Invitrogen which has a buffer including the dNTP's, magnesium and the protein necessary to allow for the Taq to work.
I will still consider the DMSO, thank you!

#4 lab rat

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Posted 24 August 2009 - 02:30 PM

No problem. I would like to add, since you're using eukaryotic DNA, that I used BSA for eukaryotic PCR. (0.5 ul/rxn.) Good luck!

lab rat
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.




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