Problems with amplification - (Jun/25/2008 )
I am having problems with PCR amplification of a 1 kb gene from genomic DNA. I am using 50 ng of Template DNA (as is routinely used in our lab) for a 25 ul reaction mix. My primers have Tm 54 and 59 C and GC(%) of 32.3 and 53.8 resp. I have tried annealing temperatures of 48 to 52 for 1 min. I get lots of primer dimers and no amplification. I am using a regular Taq buffer containing MgCl2. I am also having problem with another PCR, wherein I am getting the product of right size, however the yield is very low. Here also, I am seeing some left over primer-dimers. The details of this amplification is given below.
Amplicon of 0.7 kb (primers have tm of 53 and 55 and I am getting best product at 50oC with no product at 48oC and lesser product at 52oC). Here also I am using genomis DNA as template (50ng). How can I improve the yield? PLEASE HELP. Thanking you in anticipation
Amplicon of 0.7 kb (primers have tm of 53 and 55 and I am getting best product at 50oC with no product at 48oC and lesser product at 52oC). Here also I am using genomis DNA as template (50ng). How can I improve the yield? PLEASE HELP. Thanking you in anticipation
u can increase the yeild by increasing the Mgcl2 conc. But keep in mind increased Mgcl2 may lead to non specific amplification as well.
so do it in little increments.
other wise u elute your desired band from the gel and reamplify using it as template.
Presently I was using buffer containing 15mM MgCl2. Today, I have set up one with 30mM MgCl2. I will let you know what results I have got. I even thought of a reamplification or band-stab PCR, however I was worried if I may be amplifying some DNA with a mutation, though with such smaller fragments, I think the chances are less. Thanks a lot for the suggestion
Amplicon of 0.7 kb (primers have tm of 53 and 55 and I am getting best product at 50oC with no product at 48oC and lesser product at 52oC). Here also I am using genomis DNA as template (50ng). How can I improve the yield? PLEASE HELP. Thanking you in anticipation
1. Your annealing time is very long. I usually use 15 seconds, and this is more than sufficient. The longer the annealing time, the more chance of non-specific amplification.
2. Primer 1 in your first reaction has a very low GC content: I'd say too low. Can you change this somehow?
3. Do you know the GC content of the amplicon? If it's high, you might need to add enhancers such as Betaine or DMSO to increase PCR efficiency.
Ginger
do you try different concentrations of DNA OR primers? This could be the cause of dimers of primers.
50ng with 10p of each
100ng with 25p of each
200ng with 50p of each.
I put on my pcrs first in 50 Ul of final volumen and after reduce to 25Ul .
Which is the method of extraction of DNA?.. It is pure?.. Perhaps if it is not in a final volume of 25ul interferes..
I think 30 seg of annealing is ok. the extension not be less than 1 min.
sorry my english is disgusting.
Amplicon of 0.7 kb (primers have tm of 53 and 55 and I am getting best product at 50oC with no product at 48oC and lesser product at 52oC). Here also I am using genomis DNA as template (50ng). How can I improve the yield? PLEASE HELP. Thanking you in anticipation
u can increase the yeild by increasing the Mgcl2 conc. But keep in mind increased Mgcl2 may lead to non specific amplification as well.
so do it in little increments.
other wise u elute your desired band from the gel and reamplify using it as template.
I think that your primers are not specific enough; that is, not long enough. Try to redesign them to give a Tm of 60-65 degrees, then use an annealing temp of 55-60. Use the old "2 degrees for a C or T, 4 degrees for aG or C" equation when calculating Tm, because it's simplest and often works best!!
Happy amplifying!!
1. Your annealing time is very long. I usually use 15 seconds, and this is more than sufficient. The longer the annealing time, the more chance of non-specific amplification.
2. Primer 1 in your first reaction has a very low GC content: I'd say too low. Can you change this somehow?
3. Do you know the GC content of the amplicon? If it's high, you might need to add enhancers such as Betaine or DMSO to increase PCR efficiency.
Ginger
[/quote]
Thanks a lot for help.
1. Regarding non-specific amplification, my problem is that I am not getting any amplification.
2. The problem with my first primer is that there are too many "A" in that area which is bringing down its GC. Therefore, I tried to increase the length (31 mer), still the GC is not improving
3. The GC of my amplicon is around 45-50%
u can increase the yeild by increasing the Mgcl2 conc. But keep in mind increased Mgcl2 may lead to non specific amplification as well.
so do it in little increments.
other wise u elute your desired band from the gel and reamplify using it as template.
[/quote]
I think that your primers are not specific enough; that is, not long enough. Try to redesign them to give a Tm of 60-65 degrees, then use an annealing temp of 55-60. Use the old "2 degrees for a C or T, 4 degrees for aG or C" equation when calculating Tm, because it's simplest and often works best!!
Happy amplifying!!
[/quote]
The problem with my primers are that they contain a lot of "A" due to which the Tm becomes very low. My primer is already 31 bases long and even if I increase it by few more bases, there is really no drastic increase in the Tm. Thanks anyway !