Science Buddies: "Ask an Expert"

Hi,
My hits for the forward sequence above my gene are:

http://www.ncbi.nlm.nih.gov/entrez/quer ... pt=GenBank

http://www.ncbi.nlm.nih.gov/entrez/quer ... pt=GenBank

http://www.ncbi.nlm.nih.gov/entrez/quer ... pt=GenBank

The hits are the same for my reverse primer as far as the first four E coli hits.

The gene I am looking at, cpdA (also known as the Icc gene), is the four hit. Here is the link to it:

http://www.ncbi.nlm.nih.gov/entrez/quer ... pt=GenBank

Thank you very much

Since the other hits are for the whole Ecoli genome i think its probably ok.
Another test you could try is "in silico PCR"
http://www.in-silico.com/PCR-RFLP/index ... scherichia
If you get a lots of products with your primer pair then maybe you need to make the primers longer to increase their specificity.

Hi y'all,

You've done a really great job designing your primers by hand and performing a blast search. I agree with Caroline; your other four hits are for the entire genome, so don't worry about it. The first gene hit that you got was cpdA, so I think your primers are ready to be ordered. Congrats!

Did you contact the teacher at the community college? Have you found access to a PCR machine yet?

Best of luck.

Linh

Hi mmb1,

You're doing a great job. I don't want to be a party pooper; but, I highly recommend that you find another set of primers. At the calculated Tm of 58C, you will probably have to pilot your PCR reaction starting at around 53C or 52C and work on up. That, in itself, is a very low Tm to start with. You risk getting a bunch of crap in your PCR reaction (random annealing). I highly suggest that you make a set of primers that calculate to around 66-68C, but try to keep your primer lengths at around 20-24nts. I am assuming that your DNA polymerase will be running optimally at 72-75C. Feel free to ask questions if I wasn't clear.

Best,

James

Greetings,

I've personally used primers with a melting temp anywhere between 50C and 70C. Usually, I shoot somewhere between 55C and 65C, so I think your set should work just fine. It's definitely worth giving it a try. If you're really worried about specificity, you could order primers that are longer in length. However, based on your blast search, cpdA was the top hit. I think it's likely that your primers will work, but if they don't you can always order more with a higher Tm. Just make sure that the product is the correct size before you clone it into your vector.

Have you checked the sequence for restriction enzyme sites? This could be important in terms of cloning strategy. If you're not sure what I mean, ask and we'll try to direct you to some useful sites.

Best of luck.
Linh

BTW We had only done how to design primers by hand. We havent got a vector yet so have not done putting restriction enzyme sites in the primers or making sure out protein of interest is cloned in frame.

mmb1 - how is it going with finding a lab to do your experiments?

Slowly but surely