Hi All,
I have started with Gateway cloning..so quite new to this. I have to convert my vector to a gateway compatible one using the vector conversion kit from Invitrogen. According to the protocol, I linearized, dephosphorylated my vector and used 50ng of the vector and 10ng of the insert (the gateway cassette containing the gateway sites which has to be inserted in my vector to make it gateway compatible) for ligation. transformed 2T1 competent cells (as indicated, for it being resistant for the antibiotic marker in the cassette) and plated on antibiotic plates selective for both my vector and insert. This is as per the protocol mentioned. But no colonies showed up for this. From this I could only say that my ligation did not work becoz of insufficient DNA in the ligation mix. SO later on I used 50ng of my vector and 40ng of the insert but still its not working. Here I want to say that the insert (cassette provided by the kit) is very dilute (5ng/ul) and only provides 50ul of that. SO I cant really afford to waste or use up the insert by increasing the amount.
Please guide me with suggestions as to why it is not working, and what possibly could be done to get this fast.
Thanks
Regards
SD
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