Problem with cloning pGEMT easy vector system - (May/31/2007 )

hai..

i hope someone can help me this out..
my PCr product size is 2.2kb then i procced to cloning.
i'm using pGEMT easy vector.this my 1st time using it..After blue/white colonies selection, i pick 23 colonies to proceed for other steps. i did PCR colonies to check for insert.out of 23, 12 colonies give positive result.but i'm not sure is that really my insert because it size was almost 3kb..After that, i try using RE to take out my insert as my alternative test. When i purified plasmid before proceed to RE digestion, my plasmid size was only appox 2.5kb with insert in it. But plamid size only withou insert was 3015bp..should be i get bigger size rite?why?can someone plz help me in this..

If I understood you right, Undigested DNA would run faster in gel as it is supercoiled. This would be the discrepancy in your sizes of your plasmid. Do a single digest or try to digest the insert out with the apprpriate enzymes and have run your pGEM-T vector side by side to verify the sizes.

hai..

i hope someone can help me this out..
my PCr product size is 2.2kb then i procced to cloning.
i'm using pGEMT easy vector.this my 1st time using it..After blue/white colonies selection, i pick 23 colonies to proceed for other steps. i did PCR colonies to check for insert.out of 23, 12 colonies give positive result.but i'm not sure is that really my insert because it size was almost 3kb..After that, i try using RE to take out my insert as my alternative test. When i purified plasmid before proceed to RE digestion, my plasmid size was only appox 2.5kb with insert in it. But plamid size only withou insert was 3015bp..should be i get bigger size rite?why?can someone plz help me in this..

I agree with Scolix.
I would suggest you to do a RE digest to release out the insert from the vector. RE digest result is far more reliable compared to colony PCR screening.
To absolutely confirm the construct, send it for sequencing.
Good luck.

Hi
pGEMT easy vec has got number of restriction enzymes use of one of those can release the insert of the plasmid. for ex., NotI this site is present upstream and downstream to your insert in the vector. Take 10ul of your plasmid and set restriction digestion reaction.I am constantly working with the vector. I never had problem with it. Did you follow the ligation reaction as prescribed by the manufacturer? If so, you would have surely cloned your stuff inside. Dont bother about colony PCR results. Go ahead with restriction digestion otherwise sequence it. All the best