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Restriction of pichia vector pPICZ alpha with Xho1 and Xba1 - (May/07/2007 )


Jux starting with the expression or the starting stages of expression u can say...started with restriction digestion of the pichia vector pPICZ alpha A vector with the enzymes Xho1 and Xba1.
The reaction conditions was as per said by the NEB enzyme protocol. After restriction ran the sample on gel and found a band at 3.6kb. My question when we cut the vector and purify it shud i go for a CIP treatment as done for TA cloning to remove the phosphate??? I did a ligation of the gene of interest with this restricted vector and the transformation in Ecoli top 10F was done, but i got white colonies when i had transformed the gene of interest with pPICZ alpha and a transformation with the restricted vector pPICZalpha only. Iam suppose to be getting white colonies only with pPICZ alpha A+ gene of interest but the results are confusing as i have got white colonies even with the restricted and linearised vector pPICZ alpha A.
Posssibilites are that my restriction digestion of the vector was incomplete. How do i solve this problem.???? please do help me its very urgent i solve this problem.

thanks a lot.


Hi all...

aybody who has worked with pichia can help me out will be too gud of u...