Ask about linker ligation - (Jul/11/2008 )

Dear all,
I do the linker ligation (myc tag) follow this protocol :
Mix 1ul of linker primer F (100uM) and 1ul of linker primer B (100uM) and 6 ul dH2O. Incubate at 80C for 5 min. Leave at RT until the tube cool down. Then add 1ul of T4 ligase buffer and 1 ul of T4 polynucleotide kinase. Incubate at 37C for 30min, then incubate at 65C for 5 min.

Ligate linker into plasmid pVenus (I cut this plasmid with XhoI and sure that the digestion is OK) (the ratio between linker and plasmid is 1:2)
using ligation mix. Incubate at 16C O/N. transform into DH5alpha...But No colony in my plate.
Do you have any suggestion with my protocol?
Please give me an advision.Thank so much

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1. I typically use 20 fold excess linker, you seem to be using 2 fold excess vector.
2. You didn't mention how much vector you are using, 100ng works best for me.
..

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thanks cellcounter,
this is my ligation reaction:
plasmid : 2 ul ( i do not measure the concentration of plasmid)
linker : 1 ul (concentration is 100uM)
ligation mix : 3 ul

What is 20 fold excess linker? Sorry, because i am the newcomer of this forum...

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In our group, we anneal the two primers in 1x ligase buffer, rather than dH20. The MgCl2 can help if you have potential secondary structures, and the salt and Tris keep everything else happy.

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1. You must measure plasmid conc and use about 100ng plasmid DNA
2. You need to add 10 or 20 times more linker molecules than plasmid molecules.
3. As Swanny suggested, it is better to use ligation buffer while annealing oligos.
4. If you are new to this technique (ligation in general); you have a reason to read basics of ligation.

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Thanks for all your advision. My problem was fixed.
Now, I try to ligate another linker (HA tag)
- i digest plasmid with BglII and SalI
- HA linker have the restriction site of BglII and BamHI
- Digest insert with BamHI and XhoI.
Then ligate together - the ratio between plasmid and insert is 1:3 and use 10 fold of linker ...but I fail...
I wonder wether the restriction site of XhoI and SalI can anneal ?
Please give me some suggestion...thanks

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XhoI recognises CTCGAG, and SalI recognises GTCGAC, so they won't anneal. If you go to the NEB website, it has a list of isoschizomers for each enzyme; look under "NEBuffer Chart".

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