a simple try would be to use a robust polymerase,,,some of the KOD polymerases are very forgiving, AS you have tried many things I suspect that your dna preparation method is leaving contaminants which look like dna but inhibit the reaction. What sort of values of OD 260/280 and 230 are you typically getting in your dna and are low values of 260/280 corresponding to your failed pcrs?

Hi Gary

For high GC-containing sequences I would advice you to use DMSO as an additive of your PCR reaction to increase stringency. Typical concentrations for a starting screening will be between 6 and 12 % of the final PCR reaction volume.

Good luck. Best

Paco

Hi all,

I wish I could use the nanodrop, but currently my lab's nanodrop was sent away to be recalibrated by the manufacturer.

I think I may have answered my own question. I did PCRs using tween with BSA, or betaine, or DMSO. My PCR gel suggest that tween and BSA works best. As I understand it, BSA works well for samples with much phenolics, which may be expected for my plant-originated fungal sequences.

Thank you.

Hi Gary,

What concentration of BSA and Tween 20 did you use?

I'm also having some trouble amplifying 16S and ITS of DNA extracts from plant roots.

My sample have a DNA concentration of around 20 ng/ml and I use the Qiagen Taq Polymerase Kit.

So far I've tried diluting 1/10, 1/50 and 1/100. I also tried adding 2 % BSA[10mg/mL] + 2% DMSO + 6 % MgCl2[25mM].

The temperature profile for the PCR I used was:

94 for 15min

_____________

l 94 for 45sec

l 52 for 60sec X40

l 72 for 60 sec

_____________

72 for 10min

Hi Simon,

You could try my PCR recipe (attached), although I expect you'll likely have to modify it to optimize for your samples. For instance, that PCR recipe may yield excessive primer dimers, so it may be beneficial to decrease the primer volume.

My thermocycler program was as follows:

(X1 Cycle)

95C for 3 minutes

________________

(X24 Cycles)

95C for 30 seconds

52C for 30 seconds

72C for 30 seconds

_________________

(X1 Cycle)

72C for 7 minutes

4C for indefinite storage

I hope this helps.

Cheers,

Gary

the percentage of Tween20 should be in 0,1-2,5% range. if the problem continues, annealing temperature may be decrease. Or, it is time to change the additive you use. The two additives that are most useful are DMSO 5-10% (v/v) and betaine(1M). however, it is advisable to adjust the denaturation, annealing and extension temperatures down by 2 C degree when using betaine to adjust conditions for the weakening of the dublex bonding interactions and enzyme stability.