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cDNA cloning problem - (Mar/12/2009 )

hi this is namitha.. im doing porject on cloning, expression and purification of GST from proteus mirabilis into E.coli. im doing from past 3 months..still im stuck with cloning itself. i dont know y am not abel to produce clones. im using pET32a vector for cloning GSt gene . the gene is abt 26k da. and pet 32 a iv been digesting it with ECO R1 and HIND III . im getting all restriction correctly and even after ligation im geting the colonies only in expected ones.the control doesnt have colonies and also the CC amp+ . then after isolation of plasmids we do pcr screening with GSt primers to confirm the presence of clones but in that step alone im not getting amplification my positive control gets amplified int hat step..but i did this trice till now. still the same result. i transformed it in BL21 E.coli cells....


if anybody knows anything regarding this plzzzzzzzzzzzzzzzzzzzzzzzzzzzzz let me know...i just have 2 more weeks to complete expression also,.. im already out of time..please some one help me out..i willl be very gratefull...

-namitha-

namitha on Mar 12 2009, 09:32 AM said:

hi this is namitha.. im doing porject on cloning, expression and purification of GST from proteus mirabilis into E.coli. im doing from past 3 months..still im stuck with cloning itself. i dont know y am not abel to produce clones. im using pET32a vector for cloning GSt gene . the gene is abt 26k da. and pet 32 a iv been digesting it with ECO R1 and HIND III . im getting all restriction correctly and even after ligation im geting the colonies only in expected ones.the control doesnt have colonies and also the CC amp+ . then after isolation of plasmids we do pcr screening with GSt primers to confirm the presence of clones but in that step alone im not getting amplification my positive control gets amplified int hat step..but i did this trice till now. still the same result. i transformed it in BL21 E.coli cells....


if anybody knows anything regarding this plzzzzzzzzzzzzzzzzzzzzzzzzzzzzz let me know...i just have 2 more weeks to complete expression also,.. im already out of time..please some one help me out..i willl be very gratefull...



Hi! How much yield did you get with the plasmid purification? Did you run the plasmids on a gel? Maybe try to cut them with a single cutting enzyme to check length and then try amplification with another primer pair that starts a bit further away from the insert.... Maybe that can help you... I keep my fingers crossed!

-isbow-