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hygromycin primers

hygromycin primers - Protocols and Methods Forum

hygromycin primers - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-07-2007, 10:18 PM
SVemuri12@gmail.com
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Default hygromycin primers



Hello,

To those who have worked with hygromycin primers for genomic DNA:

I am trying to do PCR amplification of my genomic DNA (WT and mutant
lines). So far, I haven't had any luck in getting the Hyg primers to
hybridize to the DNA templates. I have observed various annealing
temperatures and wondering if these are the annealing temperatures
that are often used in hybridization of these primers.

Forward primer:
5' AGC TGC GCC GAT GGT TTC TAC AA 3'
Tm = 61.70 deg Cel

Reverse primer:
5' ATC GCC TCG CTC CAG TCA ATG 3'
Tm = 58.84 deg Cel

For the PCR reactions I have used annealing temperatures ranging from
58 to 61 deg Cel. But none of these have shown PCR products. Should I
use a higher annealing temperature? When I manually calculate the
primer Tm using formula :
Tm = 4 (G+C) + 2(A+T), I get
Tm = 70 deg Cel forward primer and Tm = 66 deg Cel reverse primer -
both which are much higher than listed on the tubes.

I can see there is DNA after I run the gel, however no hybridzation
occurs. So, DNA is there. Are there any suggestions on making these
Hyg primers hybridize to my DNA templates?

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  #2  
Old 10-09-2007, 10:16 PM
WS
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Default hygromycin primers

Dear [Only registered users see links. ]

Basically, I would not give too much on calculated annealing
temperatures. I'd simply check the reaction in a gradient cycler (if
possible, else just run a set of samples prepared from one Rxn mix
with different Ta) where you may get an idea about at which annealing
temperatures your product looks best. If you have that HygR gene /
cDNA somewhere in a plasmid, too, you might use that as well for your
studies (in parallel) at a sub-maximal dilution (if not yet, simply
clone your PCR product into e.g. a TA vector). Will be a nice positive
control for the PCR system itself, including the primers.

Best regards. Wo

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  #3  
Old 10-11-2007, 11:06 AM
corona
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Default hygromycin primers



1. Unbalanced Tm and primer lengths doesn't help. Primers should end in one
or more G or C's.
2. Make sure the genomic DNA is clean.
3. I hope your using controls to ensure everything that should work is
working properly.


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