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Problem with Ligation

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Old 03-08-2013, 03:00 PM
Pipette Filler
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Exclamation Problem with Ligation

Hello, I have been trying to do a ligation for quite some time now with little luck, hoping someone can help me out.

I have a 13kb Vector that I want to ligate to a 2.6kb insert. Both had one too many cut sites (BstBI) but it appears to be the only enzyme I can successfully use to get the plasmid I want. I had to do partial digestions with different concentrations of BstBi at different times in order to get the fragment size followed by running on 1% agarose gel and gel extraction using Fermentas gel purification kit (says it can extract plasmids up to 20kb with at least 60% efficiency over 10kb)
The 260/280 and 260/230 ratios of he gel extracted fragments were a bit low so I did a subsequent ETOH precipitation and resuspended in 15uL of DNase RNase free ddH2O and got concentrations of: vector: 16.67ng/uL and insert: 8.22ng/uL using a nanodrop.
I am using antarctic phosphatase to dephosphorylate my vector for 1hr a 37 followed by 20m at 65 to inactivate. (The protocol says use 1mg of cut vecotr DNA, But I only have about 200ng total - not sure if this will make a difference downstream)
I am using T4 DNA ligase (invitrogen) to ligate my vector and insert.
50ng of vector, 30ng of insert (3:1 molar ratio) 1 hr at 23degrees C
I am using Max Efficiency DH5alpha competent cells for transformation (Invitrogen) Transformation includes adding 2uL of diluted ligation, flicking gently to mix, 30m on ice, 45s at 42degree C water bath, ice 2 m, add 900uL SOC media, 37degree C 250RPM shaker for 1 hr, plate on Kan (50mg/mL) Plates O/N at 37degrees C.

Controls: pUC19 DNA : got colonies
Uncut vector without ligase: many colonies (telling me by vector does have the kan resistance gene)
Cut Vector with NO insert (+) ligase: no colonies

I plated my ligation at different concentrations, the highest being 600uL (spun down and plated in a volume of 100uL) of the 1000uL I had and the lowest being 20uL. On this plate I got one colony, which i have not tested yet, but I expected to get many colonies.

P.S. I have done this ligation 2 times prior to this time using subcloning efficiency DH5a cells -- these experiments yielded no colonies at all.

Does anyone have any suggestions as to where I may have went wrong?

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