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I am trying to clone my 1.5kb gene into an insect vector known as pIB-V5His but having problems over the last 6 months. I run a typical restriction digest using SpeI and SacII for my insert and vector as follows:
10uL insert
2uL NEB Buffer 4
2uL BSA (10x)
4uL dH2O
1uL Spe I
1uL Sac II
6uL pIBV5-His vector
2uL NEB Buffer 4
8uL BSA (10x)
4uL dH2O
1uL Spe I
1uL Sac II
37C O/N digestion
I load 10uL of each digest onto a 1% agarose gel (for gel extraction) and I use remaining 10uL as unpurified form in a ligation
Ligation for both Gel extracted and unpurified are as follows:
12uL insert
4uL pIBV5-His vector
2uL T4 ligase buffer
2uL T4 ligase enzyme
I also run a -ve control as follows:
4uL vector
12uL dH2O
2uL T4 ligase buffer
2uL T4 ligase enzyme
Ligation occurs at 16C O/N
I then transform into XL2 Blue cells. I do get clones but my vector transformation has more colonies than my ligation transformation. In fact I should see more clones in my ligation
I go on to pick clones from my ligation and extract the DNA using Qiagen's mini prep protocol
I do a redigestion in the hope to cleave the supposed "construct" to yield in cut vector and insert. I load onto a 1% agarose gel BUT I don't seen my insert band!!
Please could someone give me some advice on what the heck is going on! What am I doing wrong?? I have wasted 6 months in trying to clone and getting VERY frustrated now
Many Thanks for your suggestions
