
#1
Posted 14 November 2012 - 01:32 PM
#2
Posted 14 November 2012 - 02:13 PM

Andreea
#3
Posted 14 November 2012 - 05:58 PM
I use the Fermentas master mix according to supplier protocol.
DreamTaq Green PCR Master Mix (2X) 25 μl
Forward primer 0.1-1.0 μM
Reverse primer 0.1-1.0 μM
Template DNA 10 pg - 1 μg
Water, nuclease-free to 50 μl
Total volume 50 μl
PCR program
Initial denaturation 95 3 min 1 cycle
Denaturation 95C 30 s
Annealing 56C 30 s
Extension 72C 20 s
35 cycles
Final extension 72C 7min
Expected size of the PCR product is 253bp.
Hope this would help !
Edited by uday, 14 November 2012 - 06:00 PM.
#4
Posted 14 November 2012 - 11:02 PM
#5
Posted 20 November 2012 - 01:01 AM
If it is possible, you could try to reamplify your low PCR product making a nested PCR (I mean, using internal primers). This usually works very well.
#6
Posted 20 November 2012 - 03:03 PM
0.5μM primers. 100 ng of template (I woudn't generally go higher than 300 ng of gDNA in reaction).
Tell me if it helped. DMSO saved few of my assays.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
'Normal' is a dryer setting. - Elizabeth Moon
#7
Posted 20 November 2012 - 11:17 PM
Just for additional info, what are you planning to do next, with the PCR product?
A gel picture of the low PCR yield would be of great help to all of us here.
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#8
Posted 10 December 2012 - 04:49 AM
i am getting a low PCR product ! I have tried reducing the annealing temp, increasing the primer and DNA template conc. too. But still quite low. Anything I should try ?
Hi,
There can be many contributing factors for getting a low PCR product. You can troubleshoot the following:
1) Check whether the primers you designed are perfect complementary to the DNA strands. Perhaps they are not specific which is one of the major reasons for low PCR product yield. Moreover they should not be self complementary or each others complement. If you designed them manually, I would suggest using a good primer design software. There are many tools available in the market. I personally prefer and use Primer Premier to design my primers and the tool has not disappointed me so far. Here is its link which I found on their website: http://www.premierbi...sign/index.html
2) You can try to reduce the volume of sample in the reaction mix if required and carry out another ethanol precipitation with the samples.
3) Try to optimize Mg2+ ion concentration and use a good proofreading enzyme with high fidelity.
All the best for your assays.
Cheers!!
Sabastian
Edited by v.sabastian, 10 December 2012 - 04:51 AM.
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