Cloning trouble......help! - (Jul/16/2007 )

Hi

I am trying to do cloning for reallly first time and have got tons of questions in my head!

Both my vector and insert have the Xba I sites so I did the digestion of insert and digestion followed by dephosphorylation of the vector and this was followed by gel extraction of the insert and dephosphorylated vector DNA using Qiagen kit! (I did the dephosphorylation of my vector at 37 degress for 1 hr using shrimp phosphatase).

After that I ligated the vector and insert DNA in the ratio of 1ul:3ul and set up ligation using T4DNA ligase. (37 degrees , 2 hrs). then did the transformations...but it did not work. I got 3-4 colonies in all the plates inclusive of the control plate!!!

Now I am wondering where exactly I might have gone wrong and what should be my strategy now?

any help will be greatly appreciated.

Thanks
rits

firstly - you can over dephosphorylate your vector. I am not sure how important this is when using SAP, but for CIP (calf alkaline phosphotase) over dephosphorylation is fatal. Do find out the concentration of your vector and insert.
Furthermore , the dephosphotase has to be deactivated. SAP can be deactivated by heat or phenol/chloroform. CIP can only effectively deactivated by phenol/chloroform.

Another point of danger is the UV exposure when excising the DNA band from the purification gels. UV is bad for DNA. So make sure you turn your UV box settings to long wavelenght (low energy) and work fast. Fast means the DNA is exposed to UV for seconds. So work fast and work quickly. If you are getting a sun tan, or smell ozone, you are working too slowly and your DNA is probably useless.

Lastly when a ratio of 1:3 is called, what is meant is that the molar ratio of vector to insert is 1:3. Not the volume. Again do find out the concentration of your fragments. The most basic method is to run a small sample of your DNA against a DNA ladder. Each band within the ladder is of known quantity. But comparison between ladder and sample band you can kind of work out the sample concentration. The better alternative is to use a nanodrop machine, a photospectrometer to calculate DNA concentration.

On a personal point, I perfer ligating my DNA at 16 degrees for a minimum of 7hrs. What you are doing should be okay, if your fragments are not massively large.

We prefer to ligate at RT for 30-60 min. Well, its also a personal preference.

Try everything that perneseblue suggested, you have to verify the amount of DNA to be used in ligation.

Good Luck !!!

Also, are you sure that your XbaI site (in the vector) is not methylated? Normally, the vector information will note this.

Hi all

thanks for your suggestions, I realise I need to work out the molar concentration of my DNA fragments becoz I did ligate them simply by volumetric ratio before!!!

although I guess I did not over dephosphorylate becoz I did heat inactivate my SAP.

Another concern for me suring this procedure has been the BUFFERS. After digestion with RE, do we have have to do ethanol precipitation of DNA becoz I guess we need to use SAP buffer with SAP!! ..or can we directly add the SAP to digestion mix followed by heat inactivation of the used RE???


thanks so much once again for your help, really appreciated.....

Rits