High molecular weight band from my PCR - (May/25/2016 )

I did PCR to get a long piece of DNA (~ 6 kb) and I always got a high molecular weight band above my correct DNA band (see attached image).

I used the LongAmp Taq DNAP from NEB. I want to ask what the reason it could be? Thanks.

Tung

Perhaps your primers anneal to something other than your target sequence. A lot depends on what it is you are amplifying from (genomic DNA? cDNA? plasmid DNA? etc) as larger templates like genomic DNA have greater potential to have off-target sequence motifs that may closely resemble your primer sequences.

In reality, non-specific PCR products are just another day in molecular biology. Happens all the time. For most all intents and purposes, what you are showing is a great looking PCR for a 6kb product.

There are ways to increase specificity that may or may not help. I am a fan of touchdown PCR for getting specific and reliable amplifications. Maybe use longer primers (up to 50 bp) for increased specificity. In general trouble shooting PCR's just involves changing as many variables as you reasonably can within your budget and time frame until something works right. What fixes situation A may not fix siutation B even though they may appear to be caused by the same problem.

You can add a bit less of the enzyme or the primers (do a trite separate), rise the temperature 1 or 2 degrees or add PCR additives like:

DMSO: Thought to reduce secondary structure that could inhibit the progress of the polymerase. Especially useful for GC rich templates. Use at a final concentration of 5-10%.

Glycerol: Similarly reduces secondary structure. Use 5-10%

Betaine monohydrate: Also acts on secondary structure formation. Use a final concentration of 1 to 3M.

BSA: Very useful for templates that may be contaminated with humic acids (e.g. environmental samples contaminated with soil) and is also reported to prevent reaction components from sticking to the tube wall. Use up to 0.8 mg/ml.

Tween-20: Can neutralize SDS left over from template DNA preparation that would inhibit the reaction. Use 0.25 to 1% final concentration.

Formamide: increases the stringency of primer annealling, resulting in less non-specific priming and increased amplification efficiency. Concentration range: 1-10%.

Tetramethyl ammonium chloride (TMAC): Similar action to formamide. Try out concentrations from 10-100mM.

7-deaza-2′-deoxyguanosine: A dGTP analogue that is especially useful for extremely GC rich templates. Success is reported with up to 83% GC. Use a 1:3 ratio of dGTP:7-deaza-2′-deoxyguanosine.

Te only way is to test empirically one at the time or mix 2 of them (for example I use betaine and TMAC for a very fastidious reaction). 

It is possible your primers are not specific enough. Or how much template do you use for PCR, whether it is the template remains?