Cloning Issues with Rest Digest/Ligation/Transformation - (Apr/09/2009 )

I need some help!

I've been trying to clone a recombinant gene with 2.1 kb fragment ligated to 100 bp fragment. I've tried several different approaches but now I have the 100 bp fragment created by Blue Heron and placed into a commercial pUC57 vector. I have tried to digest out a mini prep with enzymes BamHI and SpeI in NEB4 from new england biolabs (after doing a sequential digestion with PCR purification between digests). Below is my set up:

DNA (>5 ug): All of my mini prep (50 uL)
BamHI: 2 uL
SpeI: 3 uL
NEB4: 10 uL
BSA(10x): 10 uL
dH2O: 25 uL

Total rxn volume: 100 uL
2 hour digestion, 300 rpm, 37 degrees

After running it on a 2% agarose gel to isolate my 100 bp fragment, I only see a faint band even with excess amount of enzyme and plasmid available for digestion.

I isolated the band, set up my ligation reaction (T4 Ligase) and transformed into Invitrogen's DH5-alpha Chem Comp cells. Grew at 32 degrees on LB-AMP plates for 2.5 days (my sequence is very unstable and grows better at 32 degrees over a longer period of time compared to 37 degrees O/N).

My plates had 1 big colony (50uL transformation volume) and 2 groups of multiple colonies (200 uL transformation volume). I picked the 1 big colony from the 50 uL plate and 1 single colony from each of the 2 groups of multiple colonies but am doubting that I'll receive postive results.

In the meantime, I've re-cultured colonies from the 100 bp insert in the pUC57 commercial vector and performed a MIDI prep in hopes that I will get a higher yield of DNA and thus more digested product. Before I run my digestion I was hoping that someone could recommend some tips that may be I am missing (ie - Enzyme concentration, overnight digestion, etc).

Thanks!

From my experience, I don't think you need that much of plasmid DNA (5ug) for RE digest. 1-1.5 ug DNA in a total rxn volume of 20ul would suffice. Before that, you may try the efficiency of the enzymes with lower amount, such as 100 ng DNA.
I also found out that BamHI and SpeI can both work at NEB#4 (http://www.neb.com/nebecomm/DoubleDigestCalculator.asp), so why do you need to do sequential digestion, instead of double. You may lose so much DNA during PCR column or EtOH precipitation.
Why do you need 300 rpm for RE digest. Just leave them at PCR thermocycler at 37C for 2 hours. Actually, for plasmid digestion 0.5 hr will be enough too.

Another important thing; You should inactivate the enzymes at 65C for 5 min following digestion. Still at thermocycler.
You may also dephosphorylate your plasmid via Antarctic Phosphatase (http://www.neb.com/nebecomm/products/productM0289.asp) to avoid self-ligation.
Takara's Mighty Mix works very good for every ligation.
Finally, you may check your ligation mix on the agarose gel, expecting a smear with a 100bp shift against receiving vector.

Good luck with ligation...

Thanks! The reason I increased my DNA concentration was because the restriction sites are so close together that I believe it results in my low yield of insert isolation post-digestion. I am re-running a double digest with NEB4 although BamHI supposedly has Star activity (non-specific cleavage of related sequences).

Does BamHI inactivate? The New England Biolabs manual says no but I will try that because I can't gel purify today so I plan to store my reaction in -20 overnight. Also, self-ligation shouldn't be a problem once I isolated out my digested fragments because the restriction sites are non-complementary.

Finally, how can I visualize a 100 bp shift? Should I up my agarose concentration? It seems unlikely that I'll be able to differentiate 100 bases (2.1 kb vs 2.2 kb) but I'm only a mere 1st year grad student who is still scrambling to catch up with all the knowledge and experience

GeneTurk on Apr 9 2009, 04:39 PM said:

From my experience, I don't think you need that much of plasmid DNA (5ug) for RE digest. 1-1.5 ug DNA in a total rxn volume of 20ul would suffice. Before that, you may try the efficiency of the enzymes with lower amount, such as 100 ng DNA.
I also found out that BamHI and SpeI can both work at NEB#4 (http://www.neb.com/nebecomm/DoubleDigestCalculator.asp), so why do you need to do sequential digestion, instead of double. You may lose so much DNA during PCR column or EtOH precipitation.
Why do you need 300 rpm for RE digest. Just leave them at PCR thermocycler at 37C for 2 hours. Actually, for plasmid digestion 0.5 hr will be enough too.

Another important thing; You should inactivate the enzymes at 65C for 5 min following digestion. Still at thermocycler.
You may also dephosphorylate your plasmid via Antarctic Phosphatase (http://www.neb.com/nebecomm/products/productM0289.asp) to avoid self-ligation.
Takara's Mighty Mix works very good for every ligation.
Finally, you may check your ligation mix on the agarose gel, expecting a smear with a 100bp shift against receiving vector.

Good luck with ligation...