No PCR products?

i'm a student trying 2 get AGL20 gene products via PCR from the plant Piper sarmentosum Roxb (betel). I'm using AGL20 primers, 5'-ccc-cat-atg-gtg-agg-ggc-aaa-act-c-3' and 5'-ccc-gga-tcc-tca-ctt-tct-tga-aga-aca-a...





problem is however, that i have not been able to produce any pcr product bands even after %26gt;20 tries (absolutely no pcr products). the genomic dna samples used for pcr have been extracted using a modified CTAB method and show quite well on gel electrophoresis. i have tried variations with the PCR mix, including increasing or decreasing template/MgCl/dNTP/Taq/primer concentrations, but none give any results. hot starts, increasing denaturation and annealing times have also been tried. the annealing temperatures that i use range from 55-65 degrees C.





I know for a fact that the plant is capable of producing PCR products becos my senior has done it, although the products have all degraded.





so what r d problems that may be involved, n solutions?

No PCR products?
You are using high GC content primers and your first primer has a major hairpin in it that likely is preventing it from hybridizing. Check it out... you have CCCC----GG-G-GGGG in that primer. You know the G's are going to bind to those C's in a hairpin. I think that first primer is your main concern, I really do. I'd redesign one that doesn't have that hairpin. Also with that GC content, you could try actually RAISING your annealing temp up to 70 or so.





Good luck, but I think you really do need to re-design that first primer.
Reply:Technical;


1. change all your tips that u use to set up the reaction.


2. Try using a different PCR machine


3. You say you changed the annealing temp. if your PCR product is large (i.e. %26gt; 2 kb) then you might need to increase the elongation time, I would leave a minute per KB, roughly.


4. can try re-ordering primers or other ingredients (NTP's). Primers may be contaminated with DNAse or NTPs might expire.. so just try and borrow the reagenst from a lab next door or something.


5. Make sure the extraction protocol does work. perhaps get a different pair of primers and amplify another region with the same genomic DNA





Theory:


check the sequence of the primers and perhaps design new ones. It migth be that they face the wrong way or something.





eventhough unlikely, some sequences may be wrong on NCBI. so worth trying a new pair of primers..





thats all i can think of..





best of luck
Reply:I don't know the template sequence so I don't know whether all of your oligo hybrizes on the template or not.





The first thing that comes into my mind is to use annealing temp 50 deg C (that's my personal despair-limit). If that doesn't work then you have a problem with either :


1. your primers (bad quality, a mix-up and what they gave you is not the correct primer(s)-rarely suppliers mess-up the oligos) or if they were lyophilized maybe you didn't dissolve them properly.Of course check again their design and orientation.


2. your polymerase might have gone bad.


3. your template: either the prep has some contaminant that kills the reaction or the sequence is somewhat different.(you might have a strain with a bit different sequence than in the database which could be a mutation or simply a different codon for the same amino acid).





Use a fresh proof-reading polymerase. This way if you have a mis-match close to the 3' of any of the primers the polymerase can chop up a bit the end and amplify the template.





Use as a positive control a different reaction (template and primers that have worked before) so that you can check for the polymerase/dNTPs


Use a second positive control: the template you have plus a set of different primers that should wotk on it. That gives you a control of the template quality. If you want re-purify some of your template if you want to be sure that there are no contaminants by ethanol precipitation or using an appropriate kit.





You can do nested PCR. Use oligos so that you amplify a bit bigger DNA fragment ( do only a few cycles) and then use your "normal" primers to amplify the desired fragment.





If you have a difficult and/or long template use a mix of polymerases e.g. Taq+Pwo

birthday flowers