Good morning everyone.

I would like to rescue the virus from clones.

Now I have 4 inserts of DNA.

DNA 1: ~4.5kb (digested by AscI/PacI)

DNA 2: ~4.7kb (digested by PacI/KpnI)

DNA 3: ~4.5kb (digested by KpnI/NaeI)

DNA 4: ~7.4kb (digested by NaeI/BamHI)

My vector has a size of ~14kb (digested by AscI/BamHI)

I did ligase by T4 DNA ligase kit with 225ng of vector and ratio vector:insert=1:3 for each insert DNA. The ligation mixture was incubated at 14oC for 20h and transformed it to DH10B by electroporation. The condition of the electrical pulse is 25uF, 2.5kV & 200Ω with 0.2cm cuvette.

After incubating the plate overnight at 37oC, I have many colonies on my plate. I confirm the positive colony by Colony PCR to check the binding between DNA1/2, 2/3 & 3/4. But I had nothing on gel after PCR.

Also, I perform 2-step ligation.

Step 1 is setup ligation DNA1/2 & DNA3/4 with the same incubation condition. I confirmed ligase mixture by gel and see the band that I need. At step 2, I added more buffer, DNA vector and T4 DNA ligase and do the same incubation condition. After transformation, I also had many colonies but after perform Colony PCR, I still had no band on gel.

What should I do?

Thank you everyone for read and reply to my trouble.

More Le Phuong Nam's questions See All
Similar questions and discussions