I am an undergraduate student working as a research assistant and this is my first research experience so I am still adjusting to all of the techniques and procedures. My project is to clone shRNA into lentiviral vector (PLKO.1 TRC) and the basic protocol is as follows:
1. Anneal Oligos: mix, incubate at 95o C then let slowly cool down to room temperature.
2. Digest PLKO.1 TRC-cloning vector with Age1 and EcoR1
3. purify the 7kb band by gel extraction
4. Ligate using T4 DNA ligase: following 3:1 ratio of insert: vector (Note: my insert is 58 bp long), incubate at 16o C overnight.
5. Transform 4-5 μL of ligation mix to competent bacteria. My lab uses DH5-α E.Coli and Stable 3's. I tried transforming in both type of competent cells but still got no colonies!
Note: We use heat-shock method to transform (heat at 42o C for 45 secs then immediately on Ice for 2-3 min). Then the mix (ligation + comp cells) is recovered at 37o C in a shaking incubator.
I really don't understand where I keep going wrong and would like some help. My P.I. is unhappy with my inability to complete such a simple procedure.
What steps can I take to troubleshoot this and which points are critical? Thank you!