I set up a PCR reaction volume of 50ul with Taq polymerase and I found distinct bands of amplicon but when I double the reaction volume to 100ul to get higher volume of amplicon I found multiple bands of different sizes.
Can anyone help me to sort out the issue?
Thanks.
Usually, you get mismatch when the PCR condition are not optimum.
You need to check that you doubled the reaction contents correctly.
Also, the PCR machine should be informed about the sample size, because if it is set to 50ul while you use 100ul the temperature will not be accurate and this is a good reason for mismatch.
Also, I think you have to use the 0.5 ml tubes instead of 0.2 ml tubes.
You could, also, use a high fidelity Taq polymerase with proofreading function.
Additionally, you can add MgCl to increase the stringency.
Finally, you can use to tubes of 50ul as long as it is good and save your time and money.
I agree with Samer regarding the temperature problem. For preparative scale amplification I never use a sample volume of more than 50 uL. Instead, I distribute the reaction mix into multiple 0.2 mL tubes in small aliquots.
To get more amplicon, I usually make four/five different 0,2 PCR tubes With The same reaction mix And same conditions! Then, I collect together all The tubes And purify PCR band on low-melting agarose gel!!
As pointed out above, either your primers are not annealing properly because of the temperature, PCR conditions or poor primer design, so you need to try changes in temperature and conditions to see if that makes any difference, if not, you may want to try new primers.
Either PCR conditions are not proper or the product is contaminated another purification will do better and also maintaining proper cycle temperature and time
I have encountered the same problem earlier. When we tend to double the reaction volume, normally we add double the amount of each component and that's the problem. Adjust the components according to their final concentration and it just works fine. or as others have suggested, set up multiple reactions of small volume and pool the samples,
good luck
I think that the issue is mispriming. Most likely scenario is that when you increased to 100ul you may not have changed the cycling parameters to allow for the increased volume. What were your cycling conditions for a 50ul reaction?
Thanks all for your valuable response. I do not have gel picture right now. I have set up PCR reaction many times with 100ul scale up in 0.2ml tubes with deep vent and pfu enzymes. This is the first time I have encountered such problem with Taq polymerase.
There could be many reasons, Deep vent and pfu do not require additional MgSO4 solution. Secondly, previously I have used kit extracted plasmid samples so there might be less contaminant than in this case where i used manual preparation of plasmid without RNAse.
@Vik- my PCR condition includes 94 degree initial heating for 4 min, denaturation at 94 for 1min, annealing at 56 degree for 1 min, extension at 72 degree for 2 min. No. of cycles 30. then ending with 72 degree for 7 min followed by hold at 4 degree.
I made all reactions in 50 ul in 0,2ml tubes!! You could try different PCR conditions as follow: 94 degree initial heating for 3 min; denaturation at 94 for 30 sec; annealing at 56 degree for 30 sec; extension at 72 degree for 30 sec if your amplicon is 500 bp / 1 min for 1000 bp / 2 min for 2000 bp.
I would say your problem is one of temperature not being consistent after increasing the volume, but your boiling and annealing steps are really long at 1 minute! I've never heard of anyone doing 100ul reactions for PCR - if you know it worked at 50ul I would just do more 50ul reactions and pool them at the end. No need to re-optimise the reaction if you just need more product.
Gaurav, I think Porter suggestion is the best thing you could do now. Your condition seems similar to that of Reiji Okazaki when he attempted to discover Okazaki fragment. Not that he used PCR at that time like you did but he kept used a minimum volume of reaction (because he found a good result with it) instead of doing a doubled volume experiment. :)
For three main reasons:
your primers may be not sequence specific (two short primers)==>Use longer primers
your primers target sequences that have been repeated in the DNA applied as template==>You shall see what you aim
the annealing temperature is not well adjusted (lower than optimum)==>Optimize AT to higher degrees
your primers anneal to somewhat similar sequences at lower temperatures==>Use Hot-Start PCR
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