4 replies to this topic

[manicd]

- Hello everyone.
- I´m trying to subclone a 760 bp insert into a histag fusion vector pQE30 (3461 bp)
- First I need to digest  pQE30BG (4184 bp) (that has another insert, beta giardin) to have my vector (pQE30).
- In order to do that I perform sequential digests with kpnI (fermentas) in his optmial buffer  (10X Buffer KpnI), ethanol precipitation protocol and then SacI (fermentas) digestion in optmial buffer (10X Buffer Ecl136II, PacI, SacI). Sequential digestion with kpnI and SacI should result in 2 fragments with: 3455 bp+ 729 bp.
- In vector preparation I used:
  - 25 µl plasmid (have a total of 50 µl)
  - 3 µl buffer sacI or kpnI
  - 2 µl sacI or kpnI enzyme
   total volume 30 µl

- 2 hours 37ºC in water bath
- after 2h I add 2 µl sacI or kpnI
- and then more 2 hours in water bath. So a total of 4 hours.
- I performed controls as follows (for later agarosis gel)
- before digestion- 1 µl plasmid
-after 2 hours- 1 µl plasmid
- after 4 hours- 1 µl plasmid

- The problem is in purification of the digested vector by agarose eletrophoresis.
- I used 12,5 µl (half) in each wells and with HindIII marker.
- Can´t get a good separation between undigested vector (nicked and supercoiled vetor DNA) and the vetor that I want to remove.
-I tried to lower percent agarose gel concentration to 0,8 %, I change eletrophoresis voltage to 50 V and aim to run the gel for as long as possible.
- I attached the agarosis gel eletrophoresis. 1- HindIII marker. 3 and 4- pQE vector
  
-Thanks.


Michael

Attached Files

•  agarosis gel pQE.bmp   399.56K   117 downloads

[phage434]

This sounds excessively careful.  You should be able to do both reactions at the same time in NEB buffer 1, for far shorter periods.  I'd suggest that if you have trouble, as you appear to be having, that the difficulty is the large fraction of your reaction volume consisting of plasmid DNA.  Often inhibitors of cutting (ethanol, Gu-HCl) are present in miniprep DNA, and can prevent cutting when present.  You can avoid this by making the DNA fraction of your reaction low (that is, have the bulk of your reaction volume pure water).  I can't see your gel, but it should be trivial to separate the cut fragment and the digested backbone.  You should have little uncut plasmid after even a half-hour digestion.

High marks on providing lots of information with your question, though!

[HOYAJM]

- In vector preparation I used:
- 25 µl plasmid (have a total of 50 µl)
- 3 µl buffer sacI or kpnI
- 2 µl sacI or kpnI enzyme
total volume 30 µl

I had the same problem and phage helped me out by pointing this out. The majority of your reaction volume is from the plasmid DNA. You could have contaminants in the plasmid prep that are inhibiting the restriction digest. When this was happening to me, I repeated the plasmid prep much more carefully and obtained a higher yield and higher purity. I repeated the digest using a larger volume (75 ul for the digest) with only 10 ul or so of the plasmid DNA.

[manicd]

Hello
Any tips for ligation procedure?
Thanks

Michael

[phage434]

Ligation problems are almost always problems with either the quality of the DNA going in, or the transformation process after, and rarely the ligation itself.  I would make sure I had highly competent cells (1e8 cfu/ug efficiency or greater), and check this experimentally with a 10-50 pg transformation of pUC19, for example.

The one thing that can go wrong during ligation is lack of ATP in the ligation buffer, due to mistreatment or too many freeze/thaws.  This problem is far less likely than poor DNA or poor competent cells.