发信人: leohawk (leohawk), 信区: Biology
标 题: Re: help on ligation problem!
发信站: The unknown SPACE (Mon Jul 23 20:20:40 2001), 转信

too long to read :)

I think it is possible pet20 have leaky expression, it may kill the cells.
in this case, I think novagen have a cell line that won't have leaky expression.

Instead of cloning disgested fragment, how about do blunt cloning using
novagen's perfect cloning kit? then cut it, at least you won't worry about the
short seq at the restriction site, after cloning,
with multiple clone sites on both side, you
have more choice to put it to pet20, though you have to worry about the problem
with reading frame.

you don't have to check the DNA concentration at all, judging from the
strength of the band on agrose gel is good enough.

in my case, ligation always give something,
check if you used the wrong plate:)


【 在 royluo (roy) 的大作中提到: 】
: Hi,
: I have problem of ligating a short sequence with only around
: 150 bp into a vector. Here is what I am doing and the results:
: (1) designing two primers with XhoI and Hind III restriction
: site overhangs for amplification of the insert fragment from a
: construct.
: (2) PCR works very well, a band between 100 & 200 is clearly seen.
: (3) Purify the PCR product with Qiagen's MiniElute PCR purification
: Kit. Agarose gel after purification shows the DNA isn't lost in the
: purification.
: (4) digest the purification product with Xho I & Hind III (from Gibco,
: and they use the same buffer, so I put the two enzyme together), for
: overnight.I digest them for overnight because the there are only 3
: flanking base pair on both ends, so longer time is favored.
: (5) at the same time, digest empty plasmid,pET20b(+) from novagen,
: with the same two enzymes. There is one problem here, the two enzyme
: restriction sites on the multiple cloning area are very close, though
: don't overlap. Only 10 base pair sits between them. Therefore, I digest
: the empty plasmid with the two enzyme also for overnight to get the
: best results.
: (6) After overnight digestion, the linearlized vector &
: insert are run on a agarose gel and then subject to Qiagen's MiniElute
: gel extraction Kit purification. The finaly volume for both vector &
: insert is 9 ul. The DNA concentration of vector can be quantified by
: UV spectrometer after 1:1000 dilution. It is around 400 ng/ul, while
: insert can not be quantified in that dilution. But a pre-made DNA
: fragment sample of different concentration but same length is run
: and compared with the sample. And it is determined that the
: concentration of insert is about 40 ng/ul. Because vector is about
: 4 kb, and insert is only around 150 bp. 1ul insert's mole amount is
: three times of that of 1ul vector.
: (7) The ligation reaction is set up first with T4 ligase and buffer
: from Gibco. the reaction volume is 15ul, while 1 ul vector & 1(or 2)ul
: insert are added. At the same time, a negative control, everything
: except the insert is set up. The three tubes are kept under Room
: Temperature for around 2 hours
: (8) The three plates all shows no colonies at all after
: transformation and plating.
: (9) Suspecting the enzyme, Amp plate or competent cells may be
: wrong, I did the ligation again with a T4 ligase and buffer from
: New England lab. The enzyme was used recently by another lab, and
: it was working normally. At the same time, 0.5 ul vector & 4 ul
: insert are used to increase the insert/vector ratio. Total reaction
: volume is 10 ul , and the tube is kept under room temperature for
: two hours according to the enzyme provider's successful experiences
: before. A negative control is done also without any insert in it.
: (10) In the transformation procedure, a non-digested pET20b(+) sample
: is applied as positive control . All the amp plates are replaced by
: reliable ones from another lab.
: (11) The results are still frustrating. The positive control gives
: out a lot of colonies,showing it is neither plate or competent
: cell's problem. But the plate with ligation product transformation
: and the negative control still have NO colonies at all!
: (12) It is strange that even negative control gives no colonies.
: Because the two restriction enzyme sites on vector are so close,
: it is possible that some vector are not digested or only on one site,
: in such situations, there will be circular plasmid existing in the
: vector sample bringing negative control plate some colonies. But the
: fact is there is no colonies at all on that plate.
: Any one has idea about it? Please bear in mind that the insert is
: very short , only 150 bp. One thing I am suspecting is that the
: primers I ordered may have defects on its ends, therefore, no
: \inserts can form desired sticky ends after digestion. But it is
: also possible that because the insert is very short, the ratio of
: vector vs.insert should be very high.


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