Clone two pieces of inserts - (Dec/11/2006 )

Hey all,

Here is something I am working on and I'd like to see your thoughts on my plan. Any suggestions would be appreciated.

I am cloning a 3.6 KB promoter + 3.3 Kb gene. I found that there is an SPH1 site in the middle. So instead of 1 big 7Kb PCR I am going to do this by parts (and they said you'd never use by parts outside of Calculus). I am running PCRs on the two parts (the SPH1 site is common to both halves after all).

These parts will be called part A and part B.

part A looks like this

Promoter-first part of gene-SPH1

PartB looks like this

XXXXX-SPH1-gene---stop

I'm going to use a PCR cleanup kit to clean both pieces seperately.

I'm then going to cut them seperately with SPH1.

The cuttings will be gel purified, and the two halves will be ligated together using T4 ligase.

From here I plan on throwing it into pGEM-T vector for sequencing, if that checks out, it will go into the GFP vector I am working with.

Any suggestions or comments would be appreciated.
Thanks

So, ig I understand correctly, you would ligate the 2 cut PCR products together and then clone it directly into the pGEM-T vector?

Haven't worked with the last one, but I fear you might get some background because your PCR-producs won't be 100% cut, so either one of the can be inserted into pGEM-T.

Isn't there any possibility of cutting your promotor+protein coding sequence into the GFP vector? Or clone them one after the other into the GFP vector?

Clone one after another? Interesting, what do you mean?

First clone your promotor, check your plasmid and then clone the reading frame.
Inserting means that you clone your sequence into a vector, in this case that your promotor or your reading frame gets cloned into pGEM-T instead of promotor+reading frame.

hi,
i am not sure wether pGEMT can clone a 7 lb frgament

well 7lb fragment is pretty heavy, if I do say so myself.

However I see no reason why a 7kb fragment cannot go into a pGEMT vector. Although I agree it will be difficult, no much about the size but the method which it is being done. I'll go by a proper ligation if insert ligation is the method being used.

i worked 3 years with pGEM-T. cloning of 7kb is no problem!
greetz