TOPO TA Problems - TOPO TA Problems (Apr/21/2009 )

Hello All,

I have been using the Invitrogen Topo TA cloning kit for sometime, intially I have not had any problems cloning PCR product sizes of 1.5Kb.

But recently I have been trying to clone a 113bp product using the kit. The product is not gel purified as the PCR bands are really thick and there are no primer dimers or non specific product.
I use just 1 microlilter of the PCR product and 1 microliter of vector for transformation. Transformation is by heat shock for 30 seconds at 42C. After which the cells grow in SOC medium for an hour after which 100microliter and 50 microliter are plated on to 2 plates containing LB with Amp, Xgal , IPTG.

After 24hrs of incubation I see a lot of white colonies, but they are all small and tiny, I did a screen for 40 samples by colony PCR using M13F and M13R primers and did not get any results, also I did plasmid prep for 20 colonies and did the PCR. But I have not got any positives. Kept the plates for 48hrs and still the colonies were small.

Not sure what is happening. Anyone with any suggestions.

thekid

thekid on Apr 21 2009, 12:43 AM said:

Hi,
I have started using this kit recently, and my product is 246 bp. i also had similar problems, Getting no colony. When i contacted invitrogen, they said, after gel extraction and addition of 3'A overhang, Measure DNA concentration and then use Insert : vector in 1:1 to max 5:1 , not more than that.

I am going to try in that way.
Thanks,

Hi,

That is a sound suggestion I think , not sure whether it will work, the problem is do we still need to do a gel elution if there is no primer dimer or non specific bands seen on the gel. Also I am giving a 10 minute final extension with Taq which will add the 3' ends I hope.

Will try and measure and use it at the ratio they suggest lets see how it goes...

Does anyone else have the same problem, and have solved it????


thekid

thekid on Apr 21 2009, 07:55 AM said:

I didn't . in my hand, it works fine. 10 min is good enough for Tail adding. i did 7 min. I just put the PCR reaction ( not even purified ) into the TOPO TA reaction . works fine. quite robust.

Regarding the tiny white colonies. I don't htink it's the real deal . Might be Amp not high or strong enough ( degraded?) to effectively select for the right transformants.

thekid on Apr 21 2009, 07:55 AM said:

I didn't . in my hand, it works fine. 10 min is good enough for Tail adding. i did 7 min. I just put the PCR reaction ( not even purified ) into the TOPO TA reaction . works fine. quite robust.

Regarding the tiny white colonies. I don't htink it's the real deal . Might be Amp not high or strong enough ( degraded?) to effectively select for the right transformants.

thekid on Apr 21 2009, 07:55 AM said:

I tried using 3:1, it did not work this time too.Got very tiny 2-3 colonies in some of the plates.
And i guess, as we are doing 10 minutes extension after PCR cycle, that might be enough. May be we can avoid the 3' A addition step. But i am not sure, that will make any diffrence.

I dont have any clue what to try next. Lets see what others say.

could this be a problem with transformation . any control to verify cell competency?

In addition, small colonies because the plate is too crowded perhaps?

I too am using this kit. I had a couple of unsuccessful attempts, after that i got too many tiny white colonies when i plated them on LB +50 microg/microl kanamycin. I replated a single colony and checked for plasmid size- this tallied with what should have been if the insert made it into the vector. My problem is when i sent the colonies for sequencing, they say the DNA quantity is too low and that the plasmids 'don't read well'. am planning to try cloning once again, if you get any good results do tell what changes u made in the protocol..

you might want to clean up your PCR products before TOPO TA cloning. my supervisor told me that NH4 in PCR buffers can kill the topo cells or affect competency. i always leave my ligation for 30mins.