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发信人: zyh (OK), 信区: Biology
标 题: My solution to this.Re: cloning problem
发信站: Unknown Space - 未名空间 (Sun Jan 16 12:59:17 2005) WWW-POST
I just did it 1.5 month ago.
Let me explain my ways,
For double disgested vector, using agrose to separate the DNA fragment and
using QIAGEN gel kit to get it.
For mutated DNA fragment, you should design the primer containing restriction
enzyme cutting size plus several NT to ensure the resricting enzymes can work
efficiently. Check this information from Appendiex of NEB catalogue.After
digestion, using DNA concentrator kit to remove enzyme, salt and small end of
DNA fragment.
Mix your vector plus matuted DNA fragment (molar ratio of 1:~10) and ligate by
invitrogen T4 ligase at rt for 2 hours. Add EDTA to stop reaction plus DW
dilution and pass through DNA concentration column. Take the effluent for
transfering to compotent E.Coli TOP10.For my case, I got> 100 colony from
0.025 mL culture (total .28 mL broth). You might get very high transformation
effeciency.
Forget phenol, ethanol, they could be harmful.
【 在 calliper (calliper) 的大作中提到: 】
: I am doing cloning of a error prone mutagenesis PCR of insert (2 kb from the
: bacterial plasmid) to KpnI/MfeI double digested vector (7 kb) and
transforming
: to E. coli TG1 host. But I did not obtained a good number of colonies. I
: changed different strategies.
:
: Firstly, I did gel extraction of both vector and inserts after digestion.
: (After PCR, I cleaned PCR by phenol once and then wizard prep resin; plasmid
: was isolated by Qiagen mini kit from E.coli; double digestion of both insert
: and vector by kpnI/MfeI (NEB) for 7-16 h; gel extraction of both; then
Bio101
: geneclean kit clean, then ligate (~500 ng each) overnight at 15C, then
: isopropanol clean it, then electroporate, but 1-4 colonies obtained) I need
a
: lot (thousands).
:
: When I heard agarose and UV may hurt ligase, I skipped the agarose gel
: extraction step. I phosphotasted the vector after double digestion, and
clean
: the digested PCR inserts and phosphotasted vector by geneclean kit and
ligated
: (~200 ng each). I got 50 colonies but the library is still too small.
:
: Please help me to figure out the problems. I have worked on this cloning for
2
: months and my advisor has no patience waiting for my clones.
:
: Thanks,
:
:
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※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 129.170.]
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