Cloning 10 Kb Fragment - (Sep/14/2004 )

Hi there,

I have been trying to clone 10Kb insert into pcDNA3.1 for months without any succes.

The insert is subcloned in pUC18 from where cut it out by SalI and purify it by gel-electrophoresis using 0.8% agarose and 1X TAE Buffer.

From gel-extraction I use the Qiagen Kit.

Ligation is done by T4-ligase from USB over night at 16°C.

The insert:vector ratio is about 3:1, 1:3 and 6:1; total amount of insert DNA is about 800 ng.

Ligation is checked by PCR using a sense primer in pcDNA3.1 and an antisense primer in insert.

Afterwords I transform XL10 GOLD E.coli from Stratagene at 37°C or at
30°C.

In either of cases I got colonies, but none of theme are positive clone:contain no insert and some appear different size also.

I have been trying for months now and I am kid of desperate!!

Thanks for your help!

Laura :(

First, have you carefully looked that when you ligate the insert and the vector that the ends are compatible?
Depending on what enzymes you use...

When you have ligated, instead of analyze with PCR, take 10µl of the ligation solution and transform into heat-shock competent cells (for example DH5alpha).
In this way you will after one day see if you have colonies. When you ligate you should also have an control, only vector without insert, this is to see if self-ligation occur.

When you transform with the cells mentioned above you follow the protocol:
10µl ligation solution to 50µl competent cells
incubate at ice for 30 min
heat-shock at 37°C for 20s
incubate on ice 2min
add 950 µl LB-medium, let grow for 1 hour at 37°C, shaking
spread out on LA-plates with the proper antibiotic, i usually plate out ~100-200 µl/plate.

Is the insert cut out with only one enzyme?

Maybe the vector should be SAP (shrimp alkaline phosphatase) treated, there are many methods to optimize a cloning strategy.

I wish you good luck!

/Nina