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Multiway ligation problems: pBS + 3 fragments: bad pattern

cloning multiway

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#1 Dianthus



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Posted 10 November 2012 - 01:47 AM

Hi everybody,

I write to ask some help.

I was fightting with horrible 3 ways ligation for 6 months.

I amplified by PCR 3 fragments (1.800 + 2.400 + 2.000 pb= Lux Operon) with differents and complementaries sticky ends. The ER are NotI , XbaI, BamHI and SmaI (blunt).
(I know that XbaI has methylation sensitivity). I run the amplification in a gel and all the fragments have the right size. I digested them (6 NTD's to end to allow ER's cut).

All ensymes are new (FastDigest Fermentas)

The pBS is digested by SmaI- NotI and dephosphorylated, and has good size and pattern. Ratios 1:1:1:1 (I tried differents and are worst)

I tried a lot of different conditions:

1.- Quick ligation (BAD)
2.- Normal T4 + fresh buffer 4ºC o/n
3.- Normal T4 + fresh buffer 16ºC o/n
4.- 65ºC 5 mins + Normal T4 +16ºC o/n
5.- Sequencial ligation : first fragments and then add pBS
6.- I tried to clone it one by one. I obtain pBS + the 2n fragment (middle of the insert, but it had the XbaI site and my cells are DH5alpha, dam +). I use it to ligation (65ºC 5 mins + Normal T4 +16ºC o/n) and I obtain a lot of transformants. I selected it by the expression of the insert (Luciferase +). Few good clones.

Then, when I made plasmid extraction, digest (SmaI -NotI) and run gel all the positives had two fragments: one the insert (about 6 Kb) BUT the pBS had about 5 Kb!!

I was thinking two options:
1.- the vector is pBS+ Middle insert + Lux operon
2.- Two pBS + Lux operon (But when I digested I should see good bands, right). In fact, I think that is not possible with ColE1 ori, right?

What's wrong?

Please I need help to create my vector. My advisor isn't molecular biologist and he can't help me.

Thanks in advance.

(Hopefully you will excuse my level of English)

#2 Curtis


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Posted 11 November 2012 - 01:50 AM

This is a very difficult ligation since you have a blunt RE too. I think you need to use more digested PCR product in your ligation reaction. In order to get higher concentration of PCR product we usually clone them into TOPO or pJET , and use the digested fragments in the next ligation. this is for two reasons, first, you are sure the ends of your fragments are completely digested, and second you get a higher concentration of pure digested product.

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