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Cloning into pIBV5-His (not working)


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#1 Tired

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Posted 08 April 2010 - 11:24 AM

Hi Bioforumers,

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

#2 phage434

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Posted 08 April 2010 - 03:10 PM

Many possible things could be wrong. It is best to take a systematic approach rather than trying things at random. First, some questions.

Where does the insert DNA come from? How is it purified?
What is the concentration of insert DNA and vector DNA? How is it purified?
What is the distance between the two sites on your vector backbone?
Do you heat kill the REs prior to ligation?
Why are you using so much BSA? Are you sure it is 10x rather than 100x, which is more typical?
How are you cutting out and purifying the gel band? What wavelength are you using for the gel imaging?




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