http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Life Science Discussion en-us sci7feed@sci7.nojunkorherepleasespam.com hourly 1 2005-05-05T12:00+00:00 http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html How are you getting on with the cloning?
The above is certainly worth checking but I bet you the problem is that the restriction enzymes are not cutting the ends off of your PCR product, and thus has nothing to do with primers being left over from the PCR. Did your new PCR primer pair work? ie. did you manage to cleave the ends off and ligate it into your target vector?
]]> Sun, 16 Aug 2009 17:41:52 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Fri, 14 Aug 2009 14:33:34 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Normal
0
false
false
false
EN-GB
X-NONE
X-NONE
MicrosoftInternetExplorer4
/* Style Definitions */
table.MsoNormalTable
{m...]]> Sat, 18 Jul 2009 16:02:41 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html
The other problem - as you h...]]> Thu, 16 Jul 2009 12:41:21 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Wed, 15 Jul 2009 10:43:49 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Wed, 15 Jul 2009 10:42:29 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Wed, 15 Jul 2009 10:39:23 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Secondly, you mentioned where the extra bases go for the PCR primers; these should be at the 5' end of each primer. You can check the New England Biolab's website to figure out what the optimum number of bases to add to the 5' end of your primers so that you can efficiently cut. It really would be easier if you could use a TA or Blunt end cloning kit. These truly make the cloning process...]]> Wed, 15 Jul 2009 10:30:32 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html I went back and PCR-ed and digested again. I tried to dephosphorylate with antarctic phosphatase after my 2nd digest. I added 5 uL buffer and 1 uL AP to the digest and incubated 15 min. 37C followed by 5 min. at 65C. Then I did a Qiagen PCR Purification to remove RE. I ran 5 uL on a gel to see how much I had and...no band.
Since I already had everything rea...]]> Wed, 15 Jul 2009 10:18:42 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html 1. Depghosphorylate the vector ends. It would reduce the large number of colonies you are getting right now.
2. Digest the PCR product and gel purify it.
If this does not work, I would TOPO clone the PCR product. You could then digest the insert with BamH1/XhoI before seetting up the ligation with the vector of your interest. ]]> Mon, 13 Jul 2009 10:37:49 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Mon, 13 Jul 2009 08:57:18 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html ie.
1. PCR
2. PCR clean up, elute in water
3. If not using a blunt cloning kit: Digest with first RE
4. Purify DNA, re-elute in water
5. Digest with second RE
6. Purify DNA, re-elute in water
7. De-phosphorylate the vector (it prevents vector-vector ligations occurin...]]> Sat, 11 Jul 2009 06:40:12 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Fri, 10 Jul 2009 10:17:16 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html I think the previous responses are all good ideas. Here are a few more thoughts:
I am sure you have double/triple checked the primer design and obviously the PCR is working, but have you revisited whether you provided a sufficient number of bases as overhang for efficient cutting? Parvoman's suggestion to go through a ligation independent cloning vector would be a work around if this is a problem. Any chance you have access to some other validated primers with BamHI and XhoI...]]> Thu, 09 Jul 2009 21:02:38 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Colony PCR is a pretty good way of screening a lot of clones but I would not recommend doing a PCR screen of your ligation reaction because it is just too heterogeneous. And I wo...]]> Wed, 08 Jul 2009 11:02:30 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html I had similar experience several years ago, and found out that the vector just ligated to my PCR primers which could not be well purified out by PCR/DNA purification kit. If you did the same, you will need to do the gel purification for both your plasmid and PCR product digestions]]> Wed, 08 Jul 2009 08:22:16 GMT http://www.scientistsolutions.com/t11756-cloning+problem_+colonies+only+have+vector.html Wed, 08 Jul 2009 08:07:36 GMT