I do PCR reaction for my gene (it's 413bp). my pcr bands are so weak. I examined different template concentration (template is FMDV cDNA, 4900 nt),increase Mgcl2 concentration, and reduce primeres but my band is so weak.
pcr protocol is:
cDNA:2micro
pcr buffer 10X:2.5
Mgcl2:1.2
dNTP: 1
Forward primer:1
Reverse primer:1
pfu:0.2
water:16.1
thermocycler program is:
94.0= 5:00=x1
94.0=0:30=x37
58.0=0:40=x37
72.0=1:00=x37
72.0=10:00=x1
please help me
- There re many reasons why this might be so
- Before increasing your Mg concentration however it is important that you know your annealing temp is correct:
- Specifically, you need to esnure that your annealing temp is ~ Tm-2C compared to your primers
- However, with some primers you need to go as low as Tm -5C (usually when target region is difficult to amplify)
- Given that you state you can already see a band albeit weak @ 58C my advice would be to repeat your PCR but also try reactions @ 57C and 56C
- Indeed, if your primer Tm is 60C you might even want to go as low as 55C (i.e. Tm-5C)
- However, reduce your annealing temp to increase your band strength in 1C intervals: That is 57C, 56C, 55C (or what ever Tm-5C corresponds to)
- Is this new DNA and are these untested primers ?
- If your DNA is a new prep then try amplifying up with housekeeping primers as a positive control, e.g. b actin or GAPDH:
- If these do not amplify then you know that the problem is target related and/or the quality of your genomic prep; specially if you have amplified down to Tm-5C
- Thus, in this situation:
Also try adding 5% DMSO to your PCR reaction: This can help with a GC rich target sequence
On balance I would say that given that you can see a specific band, dropping your annealing temp by 1-3C; titrating your starting template; incraesing your primer conc. and adding 5% DMSO is likely to be the best way to get a stronger band
How much PCR product are you loading on your gel? 413 bp is smaller, but not a tiny product. Does it look faint compared to the band from your positive control?
@Laurence Stuart Dawkins-Hall thanks for your answer. I reduced the cDNA concentration, but it's didn't work(no band in gel). I reduced annealing temperature too, but it doesn't change result. I will try DMSO and other your comments. Thanks a lot.
Hi Parvin,
May I ask what PCR kit you're using? Also, what are your final concentrations of your reagents in your master mix? What are your nanodrop readings (260/280 and 260/230 ratios). If you're doing it right, 3uL should be plenty of PCR product for a gel.
What do you mean, "this kit doesn't famous"? I'm looking for catalog numbers for your reagents. What are your final concentrations of each reagent in your 25uL reaction?
Also, I'm wondering what your nanodrop readings are for your template DNA, not your product.
I missed the detail that you are using cDNA. Do you get good strong bands when you use primers for a housekeeping gene like actin? It's possible that your cells do not express your gene.
What are your reaction conditions and gel percentage? For bands that size, I usually run at 80V for 1 hour in 1% agarose gel. Also, I suggest using EZ-Vision® Dye-as-Loading Buffer, 6X). It uses the same signal as Ethidium Bromide. Also, have you included a positive control (using a sample that has worked before).
When your bands of PCR are faint, this reflects inadequate amplification of your target DNA. To me, reducing your annealing temperature may enhance better amplification but this reduction in (Ta) should not be leading to appearance of non-target bands.
Other solutions would rather include increasing your MgCl2 conc. and/ or # of cycles. You can also check self complementarity of your primers using software like GeneRunner.
Best wishes
Katie A Burnette yes I get strong band when use other primer for other gene.
Jack Chen I run at 100V for 45 min in 1% agarose gel . yes I run positive contol too.
Justin Pater HI Justin.
catalog number is: YT1603
final concentrations of each reagent in 25uL reaction
mgcl2=2.4mM
dntp=0.4mM
buffer=1X
primers=10 pM
If your product is GC rich, try 3-5% DMSO. Also, the elongation time is too important, especially in case of less than 500bp amplification! Extended elongation times produce primer extension of single strands depleting the available dNTPs in your reaction. If you are using genomic DNA as a template you may re-suspend it in TE or any other buffer, it can change the salt concentration when you put it in the PCR reaction, You can elute with water after purification.
Just use Tm, Templet, Primer and MgCl2 gradient PCR, You will get your faverable condition for PCR.
Best
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