9 replies to this topic

[eleana]

Hello,
    I have tried to do a pcr for like 1 month now and has been unsuccessful. In one protocol (below), I got a product but I have since tried the same protocol with no product. Who can suggest the reason for this and how I can rectify. The protocol:

0.4 ul of DNA (miniprep)
1.0 ul each of forward and reverse primers
7.0 ul of dNTP
5.0 ul of buffer
0.6ul of Deep vent polymerase
35.0 ul H20
I also tried Taq pol. but to no avail.
Can anyone give me suggestions? I am trying to tag a gene by pcr. thanks.

[ranvi]

try to change the kit sometimes if Dntp or the polymerase is not good then you wont get any product esp the polymerase

[Adrian K]

Tried gradient PCR? More DNA? Degraded primers?

[ranvi]

try the stratagene kit for pCR usually should work better

Edited by ranvi, 22 September 2011 - 08:45 AM.

[allynspear]

First of all, when you are describing a reaction set up, you need to specify concentrations, not volumes.  For instance, if you are using a standard 10mM dNTP stock, then 7 uL of 10mM dNTPs is way too much.  Usually 1 uL of a 10mM dNTP stock in a 50 uL reaction (0.2 mM dNTP final concentration) is used.  The same with primers.  Each polymerase is different, but a typical range would be 1 uL of a 10mM primer stock in a 50 uL reaction (again, 0.2 mM final concentration).  Lastly, your template (miniprep DNA) should be at a final concentration between 0.1 nM and 0.5 nM, which means that for an average 3000 bp plasmid, you would include about 50 ng of DNA in a 50 uL reaction.

All of these concentrations can affect how well your PCR works.  Lastly, your PCR cycling conditions are also critical.  As Adrian K suggested, you could try gradient PCR to find a good annealing temp, but in case you don't have a gradient PCR machine, I would just try this PCR program:

95 degrees, 2 minutes
----------------------------
35 cycles of:
95 degrees, 30 seconds
50 degrees, 30 seconds
72 degrees, 1 minute/kb of PCR target
-------------------------------------------------
72 degrees, 10 minutes
4 degrees, infinite

This program may generate some non-specific products, but you should at least get the band you are looking for.  If you need to get rid of backgroud, you can always try raising the annealing temperature (from 50 to 55 or 60) AFTER you can actually get a product.

Best of Luck.

[PostDocTrauma]

as above, you need to work on final volume concentrations and troubleshoot from there.

a gradient pcr is a good place to start.

who designed the primers? have you checked they synthesised the correct one? read the sticky label vs the one you have ordered.

[eleana]

allynspear, on 22 September 2011 - 11:17 AM, said:

First of all, when you are describing a reaction set up, you need to specify concentrations, not volumes.  For instance, if you are using a standard 10mM dNTP stock, then 7 uL of 10mM dNTPs is way too much.  Usually 1 uL of a 10mM dNTP stock in a 50 uL reaction (0.2 mM dNTP final concentration) is used.  The same with primers.  Each polymerase is different, but a typical range would be 1 uL of a 10mM primer stock in a 50 uL reaction (again, 0.2 mM final concentration).  Lastly, your template (miniprep DNA) should be at a final concentration between 0.1 nM and 0.5 nM, which means that for an average 3000 bp plasmid, you would include about 50 ng of DNA in a 50 uL reaction.

All of these concentrations can affect how well your PCR works.  Lastly, your PCR cycling conditions are also critical.  As Adrian K suggested, you could try gradient PCR to find a good annealing temp, but in case you don't have a gradient PCR machine, I would just try this PCR program:

95 degrees, 2 minutes
----------------------------
35 cycles of:
95 degrees, 30 seconds
50 degrees, 30 seconds
72 degrees, 1 minute/kb of PCR target
-------------------------------------------------
72 degrees, 10 minutes
4 degrees, infinite

This program may generate some non-specific products, but you should at least get the band you are looking for.  If you need to get rid of backgroud, you can always try raising the annealing temperature (from 50 to 55 or 60) AFTER you can actually get a product.

Best of Luck.

Thanks. The conc. I'm using is 7.0 ul of 2mM dNTP. Do you think the amt. of buffer is OK (the buffer came with the polymerase)?

[allynspear]

eleana, on 22 September 2011 - 01:09 PM, said:

Thanks. The conc. I'm using is 7.0 ul of 2mM dNTP. Do you think the amt. of buffer is OK (the buffer came with the polymerase)?

All PCR polymerases come with a buffer that is optimized for that particular enzyme.  If the buffer you have is 10X, then use 5 uL in a 50 uL reaction, if it is 5X use 10 uL.  But the PCR buffer shouldn't need alterations.  The only issue here is that some PCR buffers come with different levels of Magnesium (Mg 2+) which can dramatically affect the reaction.  Check to see if your buffer comes with Magnesium pre-added and at what concentration.  The standard for most enzymes is around 2 mM Mg, but many PCR polymerases come with an added tube of MgCl2, for you supplement your reaction.  Vent polymerase can often require optimization of Magnesium concentrations, and usually the range tested is simply 2 mM, 4 mM, and 6 mM MgCl2.  This is one reason that I do not use Vent polymerase that often.  My polymerases of choice are "GoTaq" for routine PCR or short products, and "Phusion" for longer PCRs or when I need proofreading activity.  I have never had to optimize Magnesium concentrations with these enzymes.  But again, be careful because different suppliers of PCR polymerases and even different catalog numbers of polymerase can have different Magnesium concentrations and you should always be aware if you are required to add more.

Best of Luck.

[mikej]

seems like everyone is missing the critical point here - you got a product once but not subsequently.

Enzyme is probably not the problem since Taq didnt work either. So something else has gone bad -

template (miniprep)
dNTPs
or primers
- buffers are rarely the problem.
Or PCR machine! check your program carefully.

Check your miniprep on a gel - unless its completely degraded there should be enough left for good primers to prime on.
Do a control reaction with some other primers on this template or a control that come with a kit - seems like everyone uses kits these days.

one of these should work!

[Rifat Rumki]

Have U ever tried to do PCR without kit (manually optimization)? I think manual optimization will work,,,, Once I faced this problem and optimized the PCR reagents manually using each reagent separately.... Are U sure about ur primers specificity?