TA cloning help - (Dec/12/2006 )

Hi I am struggling with my TA cloning. I PCR amplified my fragment from the vector and then tried to clong it into TA vector via single A overhang using TA cloning kit (Invitrogen). Here I used 4ul of insert and 1 TA vector. After ligation, I did transformation into Top10 cell. I appled 3ul of ligation product to transform 50ul cell. But the result was not ideal. Although I did get some white colonies on the plate, after the colony PCR, I didnot see any amplicon on the gel. I m just asking about the following questions:

1 Does anyone think that the colony PCR is successful? (I picked up the white colonies and dipped into 50ul water then boiled on 100C then spun down the cell debris and took upper layer which should contain DNA as template and I used 16 cycle PCR and the positive control which worked fine.)

2 Does anyone think that it is better to to 1ml culture of the colonies and minipreps DNA and do digestion of the vector, which might be a better way to select positive clone.

3 I m also wondering that if the white colonies appeared, whether they are positive clones or not. If not 100%, what is the reason to explain it. Would this be caused by the PCR amplification of my fragment?

4 Is it worth doing the PCR again using High fid Taq polymerase and treat with normal Taq to add single A?

Could anyone be patient to read through it
A great thank would be given....

Normally the TOPO TA cloning stuff is working fine so I would not worry about the cloning itself; I'd rather think that your colony PCR was not ideal. 16 cycles seems to be pretty low though your positiv control worked but I guess you do not add the same template amount in every tube. So definitley increase cycle number.

If you repeat your colony PCR you can just pick the colony, dip it into your appropriate PCR mix and afterwards inoculate 1-2 ml media to do a miniprep.
Í bet you have plenty of positiv colonies but you did not detect it with the colony PCR.

Good luck,
Cheers

Thanks a lot Bomber:

Yes I m aware that the cycle is not enough, so I increased the cycle up to 22 to see whether there will be an induction of any product, but unfortunately, I still did not get anything amplified. So I m now going back again from the first PCR to amplify the fragment.....and to see whether the result will change..

Colony PCR should routinely be run at 35-40 cycles. It is rare to see product before about 25-30. The most common problem is too many cells. You should pick a small part of a colony with a pipet tip into 50 ul of water, vortex, and then use 0.5 ul in a 10 ul PCR reaction. Don't forget to run an initial cycle of 10 minutes at 95C to lyse the cells.

So if the colony PCR dosen't work, would u think it is better to do the minipreps and digestion, which might be able to give a ideal result

Better, perhaps in theory, but too painful for words in practice. Not to mention expensive and time consuming.

minipreps take about 3 days to complete... 2 days if you push yourself. It takes a really long time to do... and takes a lot of work and attention to actually run the protocol and subsequent restriction enzyme digest

Colony PCR in contrast is very much faster to do and while the machine is running, you are free to do other stuff.

Already mentioned the minimum number of cycles is around 32. I do 34 cycles.

your colonies must not be old. Nice young smallish colonies are the best. About 1mm in diameter. Don't over grow the colony as old e coli produce lots of liposaccarides which inhibit PCR. For a 10ul volume I also add 0.25 50mM MgCl2.

But then again, you are tried to wasting time, then you could do the minipreps. It is a long but safe option.

i do my colony pcrs a different way: pick colony, dip on another selective plate to recover colony, then resuspend the rest of the colony in the pcr mix (10myl). i do 25 pcr cycles. this protocol has never let me down! i use ampliTaq Gold 2x PCR mastermix
just give it a shot!
greetz
and i think its the fastest protocol you can use. takes only ~4hrs to recover colony when the recovery plate is immediately incubated @37degC

what kit do u use for colony pcr???is it expensive?

i see a
-96 well tissue culture plate (greiner)
-96 well PCR plate (Starlabs)
-a 12-multichanel pipette
-home made taq
-home made taq buffer
-home made 50mM MgCl2 solution