发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: Ligation Problem
发信站: The unknown SPACE (Wed Jun 26 17:49:40 2002), 站内信件

with my experience, the suggestion is to check the PCR product
first. i would rather not digest the PCR product, actually, it
is better if you can subclone it first. for double digest with
NheI and XhoI, if you are using NEB buffer, use buffer2. digest
the insert and vector for enough time, and make sure your enzymes
are good (i think so from your description). unless the cloned
gene is harmful to the bacteria host, the cloning problem often
come from incomplete digest for such cloning strategy. i would
digest enough insert and gel purify, ethanol precipitate, and
use it up for the ligation. set up the molar ratio to about 10:1
to 20:1, so do not use too much vector. it seems that you do
not even have background problem, which usually comes from
the vector per se, so would you like to set up a postive control
for ligation (single digest of the vector) and transformation?
heat shock is helpful for sticky end, 4C or 12C does not matter.
i always dissolve the gel purified insert into small volume of
ddH2O and run a gel to check the concentration. and then mix
enough insert with small amount of vector, heat shock, cool,
add buffer and ligase, 16C O/N. it works for me for a long time.
good luck.
【 在 sensor (apple) 的大作中提到: 】
: OK,
: My gene is 1.7 kb, restriction sites: NdeI, KpnI, NheI, XhoI and BamHI. I used
: pET 19b ( Novagen) to clone the gene from E coli genomic DNA. I have made
: several mutations of the gene with PCR mutagenesis. However, three of them
: never work.
: the following is my whole process of mutagenesis:
: 1. two-stage PCR mutagenesis (Mega-primer method)
: 2. Gel extraction of PCR products with Qiagen Gel extraction kit
: 3. Digest whole PCR product and CV (1ug)with NheI and XhoI at 37 degree for 3
: - 3.5 hours. run agarose gel. I saw the CV and PCR product were digested.
: 4. Gel extraction of CV and Insert (elute with 50 ul water).
: 5. dry up CV and insert
: 6. dissolve cv and insert in 7 ul water
: 7. get 2ul insert and 1 ul cv to run a ligation gel to make sure there are cv
: and insert and estimate relative amount of cv and insert.
: 8. add cv into 5 ul insert and water if necessary.
: 9. heat shock 42 degree 5 min and on ice 2 min
: 10. add ligation buffer 1ul and ligase 1 unit
: 11. incubate in 4 degree overnight
: 12. transform BL21 DE3 (I also tried XL2 Blue) by electroporation
: 13. incubate in non-amp culture for 1 hour
: 14. plate the cells on 50ug/ml AMP plate
: 15. incubate 37 degree overnight.
: Marble, please give me some advice. Thank you very much!
: ~{!>~} ~{TZ~} Marble (~{P!J/M78g8g~}) ~{5D4sWwVPLa5=~}: ~{!?~}
: : be specific and tell us more detail about the experiment.
: : ~{!>~} ~{TZ~} sensor (apple) ~{5D4sWwVPLa5=~}: ~{!?~}
: But,
: plates. I
: give me


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