pcr-product cloning problems - cloning of microsatellites (Dec/12/2006 )

hello forum,

i have problem with cloning of my PCR product(microsatellites). i see lot of concatamers from my product by doing PCR of my fully grown colonies. i dont get single desired band but i get many bands. i have to clone the microsatellites of cotton. i am using the TOPO-TA cloning kit. i tried with different vectors such as 2.1, 8GW, 4. i take 2ul of the product, 2ul water, 1ul salt, 1ul vector as suggested in the manual.

i incubate 30min at room temperature for ligation.

i add 2ul of the transformed mix to the thawed chemically competent cells

incubate the cells on ice for another 30 min.

heat shock at 42C for 30seconds.

transfer the samples on to ice, add 250ul of SOC medium

keep it on shaker for 2hrs for the cell growth.

i streak the cells on LB plates(having respective antibiotic resistance) and grow them overnight.

next day i pick the colonies and do PCR reaction using the M13 primers.

i run the samples on 3% agarose at 200v for an hour.

the result will be lot of bands in the gel instead of a single desired band.

[/b]this problem persist with only the cotton DNA but not with other plant DNA samples.

please suggest me ; so as to get rid of concatamers and to get only desired single band.

this is my thesis prooject. i am burning out on this problem.

Are you getting a clean single band from your initial PCR reaction? What enzyme are you using for that reaction?

I would heat shock for 45-50 seconds. 30 s is definitely on the low end.

Tell us more detail about your PCR protocol. I'm guessing you have too many cells in your colony PCR reaction, and likely too low an annealing temperature. The gel sounds really weird. A 3% gel should be used for very short fragments -- perhaps these are? 200 volts sounds high, unless this is a large format gel.

The only way you should see multiple bands is if you have a mixed population of cells or (rarely) two plasmids in a single cell. You could try streaking out your colonies on a plate and repicking them. How close are the colonies on your plate?

Are you getting a clean single band from your initial PCR reaction? What enzyme are you using for that reaction?
i am getting single bands from my initial pcr reaction. i am using normal taq DNA polymerase from NEB.

the PCR protocol for my colony pcr is

95-3min
94-1min(40cycles)
50-1min
72-2min
72-5min
hold at 4C

yeah these are short fragments ranging from 200-700bp. i use a large format gel which has a 104 wells in it.

the colonies are pretty close and colonies are small in size comparatively looking like satellite colonies.

I'd be using a 1.5 % gel for that size range. The annealing temperature of your PCR reaction is quite low. I'd try to raise it, either by trying a higher annealing temperature with these primers (try 55) or by redesigning the primers if necessary. A 50C annealing temperature will pick up who knows what junk in a colony PCR reaction. You can probably move to 30s denature and anneal during the cycling to save yourself a bit of time. If you still have problems, let's look at the primers.

Hi gopinath,

Your annealing temp. of 50C is definitely very low.
Start from 55C and slowly increase. Try to keep annealing temp. ~ 5C below your primer Tm (although this maynot be the most crucial thing).
But use 55C as annealing temp.

Also if your products are in the 200-700bp range, why are you elongating for 2min??

this could also give you longer erroneous products, that arise due to false priming.

In short:
reduce elongation to 1min.
increase annealing temp to 55C.
30 cycles is more than enough.

good luck!