My culture is Lactobaccilus delbrueckii NCDC 253 which is claimed exact to Lactobaccilus delbrueckii ATCC 11842 but after gradient PCR for beta-galactosidase gene (3.2kb) I am not getting any band. I have isolated the genomic DNA both by kit and manually. My Tm forward is 54 and Reverse is 59. I have tried a range from 50-70. Please assist me with this.
If you could provide a little details about your PCR, then maybe someone including me can help you.. Best of luck
Hi Bapi,
I once had similar problem before, but when when I varied the concentration of my DNA I got my band. I suggest you try to calculate the best annealing temperature then you do several PCR with varied DNA concentration. It is a known fact that when DNA concentration is high the double helix might not unwound properly to enable your polymerase act.
I will also suggest you also try to use a different DNA polymerase to check if the problem is with the enzyme. I assume you are using a master mix reaction where you may not need to optimize other parameters.
Best Wishes
Hello Bapi,
If u give details about your pcr reaction mix and time details it will be easy for trouble shooting. Does your +ive control gave amplification (if you are using one). Are using a high fidelity polymerase since your product size is little high?
Thank you all for your suggestions.
My reaction details are
94o C - 5min
94o C - 1min
50-70o C - 50 sec
72o C - 2min
72o C - 8min
4oC - .....
Genomic DNA concentration used 40-50ng.
Also kindly note that I have even tried with three different MgCl2 concentration besides changing the complete PCR mixture (including DNA polymerase). I am yet to try with high fidelity polymerase.
I think you should try with high Fidelity as suggested by Nithin, since your product is large
HIII
yes you should try high fidelity (HF) enzyme,bec. normal taq can incorporate errors in your PCR, and efficiency of amplyfying large product i.e more thn 1kbp decreases.
and extnesion is v less, usually for larger product you should put more extension time, like 3min or 3.5min depending on product size and enzyme used for amplification. for example taq polymerase effiency is 1min/kbp , so if you pcr product is 3.2kbp, your a extension should be 3mins or 3.5min.
your MgCl2 optimisation and dNTP should go hand in hand . if nothings works you have to check your primer.
You can refer before starting any experiment .
all the best
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