2 replies to this topic

[rabos]

hi,
i am using  insTA CLONE kit( fermentas). after ligation my insert(1.4 kb, gel puirified) into vector - the ligated product is showing increased size  3-4 bands! ( including correct one - faintest band)- after transformation and plating- 20ul and 50 ul in amp/iptg/xgal plates- i am only getting white colonies -no blue ones( altough colony obtained are proprotionate in control , 20ul and 50ul plates . colony pcr from various colonies is -ve!
help....

[appleyun]

rabos, on Jul 25 2009, 02:37 AM, said:

hi,
i am using  insTA CLONE kit( fermentas). after ligation my insert(1.4 kb, gel puirified) into vector - the ligated product is showing increased size  3-4 bands! ( including correct one - faintest band)- after transformation and plating- 20ul and 50 ul in amp/iptg/xgal plates- i am only getting white colonies -no blue ones( altough colony obtained are proprotionate in control , 20ul and 50ul plates . colony pcr from various colonies is -ve!
help....

Hi,
I am using INSTA cloning kit (fermentas) too. After colony PCR, the insert size is 2.5kb (1kb more than the supposedly 1.5kb.). Yeah,I only got white colonies. I guess I experienced the same thing as yours.  Colony PCR of most of the white clones are negative. Really strange. Wonder what happen! PLs help us. Thanks..

Cheers,
appleyun

[malli]

Dear frnds

I am cloning pcr product 450bp into TA Pcr2.1 3.9kb vector.......i am getting both blue and white colonies.....after colony PCR with M13 Forward + my primer reverse i am getting exact size 450bp for some samples.......for other set i am getting strong band of small size 250bp.......how come a nonspecific band is coming.....

please clarify me
thanks