Normal amplification of the particular gene with the same set of primers is routine work for me. But this is how it all got ruined.
I am amplifying a set of reaction for sequencing but I am always left with secondary bands in accordance to my desired band.
Note: I dont want to gel elute the band, instead I want the PCR to be optimized
Fig 1. I do normal amplification of the 1.5 kb fragment with amplitaq dna poly (invitrogen),
PCR mix: 10x taq buffer (without mgcl2) -- 5 µl
dNTP mix (10 mM, 2.5 mM each) -- 1.5 µ
Forward primer µl -- 1 µl
reverse primer µl-- 1 µl
mgcl2 (50 mM) -- 3 µl
amplitaq pol (5U/µl) -- 0.6 µl
DNA (30ng) -- 2 µl
vol upto 50 µl
PCR condition :
94 ------------- 3 min
94 ------------- 1 min
50 ------------- 30 sec
72 ------------- 1 min 30 sec
72 ------------- 10 min
hold 4 deg.
Fig 2. To remove the secondary bands I intend to do a gradient PCR this time. (50-60 deg). The gradient PCR was done following the same pcr reaction mixture in 8 replicates. But this time again i Encounter the secondary bands.
PCR mix: Same as above.
PCR condition: 94 -----------3min
94-------------- 1 min
50-60 deg -------- 30 sec
72 -------------- 1 min 30 sec
72 -------------- 10 min
hold at 4 deg
Result: The sample amplification of my 1.5 kb band accompanied with secondary bands at all the anneal temp.
Fig 3. Two PCR products from the above reaction are column purified with Bangalore Genei PCR product purification kit.
Fig 4. PCR amplification again done with increased annealing temperature (59 deg). But the same problem persists. Non specific bands are again accompanied by the desired band of 1.5 kb.
PCR mix: same as above.
PCR condition: 94 ------------- 3 min
94 -------------- 1 min
59 -------------- 30 sec
72 --------------- 1 min 30 sec
72 --------------- 10 min
hold 4 deg
Kindly help me get rid of the secondary larger fragments in my PCR.
Hi Rajal, I've had a look on your gel pics and whithout knowing any details of your template DNA I believe it is plasmid and for me it looks like it is the plamid template what you are detecting here. The upper band's intensities pretty much correlate with the amount of amplicons and those "faint pure bands" may not be pure but contain less template, resulting in less amplicon. The upper two distinct bands just may be two DNA forms (e.g. covalently bonded and supercoiled) of one and the same plasmid. What are you thinking? Best, Robert
You should decrease the concentration of DNA after proper quantification.........................
I would try:
1) Less template (e.g. reduce 10-fold to 6 ng).
2) Shorter extension time (maybe try 1 min or 45 sec/cycle at 72C)
3) Reduce primer concentration - you don't say what concentration you're using but I would reduce to 0.1 uM if you're using more than that.
Touch-down PCR and optimising the annealing are likely to help if you have shorter products than your desired band (e.g. bands at 500 bp) but your secondary products are too big, not too small. Similarly the column purification will purify anything >50 bp efficiently. I agree with Robert its possible you are seeing plasmid, but I wouldn't expect circular plasmids to run as bands that discrete on a gel.
Guido: I agree with you and Pieter that TD-PCR should improve specificity and thinking about it you may have a point, but it hasn't been my experience that touch down has helped remove non-specific bands that are larger-than-desired. That said, Rajal has already tried optimising the annealing temperature so I don't see why touch down would help.
On the other hand, seeing as this PCR is normally routine and isn't behaving this time round, its almost certainly worth replacing the primers and (if possible) template in case something has degraded (happens astonishingly fast with a few freeze-thaw cycles).
Hi!
After watching your gel images I felt you should reduce your DNA, take 10 nano grams, Try Ta-54 C, it should work.
Good Luck!!
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