pBluescript ligation issues - pBS + thymidine kinase ligation (Aug/25/2005 )
I am currently attempting to insert a thymidine kinase cassette (2kb) into pBluescript (3kb), with no success. 
This is my protocol
1. Digest 2.5ug of the plasmids with BamHI and NotI for 1 hour
2. Run entire reaction on a 0.7% agarose gel and extract DNA from appropriate bands
3. I then set up my ligation as follows:
1uL TK (insert), 3uL pBS (vector), 1uL T4 ligase, 5uL 2XLigation buffer
4. Ligate at room temperature overnight
This is the method recommended by my PI. I have also tried various suggestions from our post doc:
-Measure exact amount of DNA from gel extracts and set up ligation in a 1:3 molar ratio (vector:insert)
-Ligate at 4C O/N
I would gladly welcome any further suggestions! I am at the end of my own troubleshooting abilities and I turn to all of you for help.
Are you digesting with Not and Bam at the same time?
If you are, don't . They have incompatable buffers (well with Promega enzymes anyway).
Digest with Bam for 6 hrs, then purify and digest with Not overnight, repurify and ligate.
Treat insert and vector the same in terms of time and digestion stratagy.
*pBluescript, who sometimes gets tired of people talking about him all the time 
*
PS: don't ligate overnight at 4C. Do it at 16C.
i read ur answer that click me i m workin on medicinal plants & one of my assay is Plasmid DNA damage. i m facing 2 problums...
at each isolation i got plasmid sharing with genomic DNA (unable to understand how to overcum this problum 
)
2nd i want complete lterature on the pBluescript (each & every thing )
can u help me these 2 problums
This is my protocol
1. Digest 2.5ug of the plasmids with BamHI and NotI for 1 hour
2. Run entire reaction on a 0.7% agarose gel and extract DNA from appropriate bands
3. I then set up my ligation as follows:
1uL TK (insert), 3uL pBS (vector), 1uL T4 ligase, 5uL 2XLigation buffer
4. Ligate at room temperature overnight
This is the method recommended by my PI. I have also tried various suggestions from our post doc:
-Measure exact amount of DNA from gel extracts and set up ligation in a 1:3 molar ratio (vector:insert)
-Ligate at 4C O/N
I would gladly welcome any further suggestions! I am at the end of my own troubleshooting abilities and I turn to all of you for help.
I would let the digestion go longer (2 - 3 hours), and I would use more insert than vector. I also do my ligations at 16C O/N (a PCR machine works well for this).
But, you've not told us anything about your host cells. Do you know they're competent?