3 replies to this topic

[Mati]

I am trying to ligate my Pcr Product into PEnter vector (invitrogen) but i cannot detect the positive insert band although there are enough colonies on the plate ?? Can anybody sujjest something to me

Thankyou

[bumbilko]

How long is your insert? You may put longer elongation step in your PCR in order to amplify it well. Are you using appropriate primers for your PCR?

You can do also restriction enzyme digestion.

[Mati]

yes of course i did restriction enzyme digestion then i run gel and check bands under U.v but i cannot detect my insert band only vector band is visible. my insert size is around 300 bp.

[Evanescence]

Hmm...
Seems like you have problem with ligation..even you have good colonies number but that all without insert..(mean there's nothing wrong with your transformation)

maybe the pcr product has no restriction site at the end of the fragment...? some people designing a fragment with few base pair (4-5bp) before RE site in their primers.. so if you did same thing make sure u digest it first...

and if u did that and no problem with digestion, maybe the ligation mixture is not good... for ligation...how long do u incubate the ligation mixture?at what temperature...try to extend it longer at 16C...Wish of luck

Evanescence