pollanen@csc.fi (03/12/91)
Hello Can anyone help me with m13 cloning I have some problems with ligation a PCR product into m13 vector (m13mp18) The size of fragments is varying from about 100bp to 300bp size in lenght. I have opened the m13 vector by pstI /HindIII double digestion and also I have digested these PCR products same way because we have planned primers so that PCR is (probably) producing pstI and HindIII sites to the ends or near of the ends of these fragments.... If anyone has tried to introduce DNA fragments into m13 vector please let me know of the problems and how did you solve those problems... Especially talking about PCR fragment cloning into m13 and also into expression vectors too... Sincerely yours Raimo Pollanen (M.S.) Pollanen@CSC.FI
fish@LINDA.LLNL.GOV (chris) (03/13/91)
RP>Can anyone help me with m13 cloning I have some problems RP>with ligation a PCR product into m13 vector (m13mp18) RP>The size of fragments is varying from about 100bp to RP>300bp size in lenght. I have opened the m13 vector RP>by pstI /HindIII double digestion and also I have RP?di>tested these PCR products same way because we have RP>planned primers so that PCR is (probably) producing RP>pstI and HindIII sites to the ends or near of the ends RP>of these fragments.... If anyone has tried to introduce RP>DNA fragments into m13 vector please let me know of RP>the problems and how did you solve those problems... RP>Especially talking about PCR fragment cloning into RP>m13 and also into expression vectors too... An alternative and efficient cloning method for PCR products was developed by Aslanidis and de Jong (see Nucleic Acids Research, 18:6069-6074). The method does not use ligation, and the cloning efficiency is extremely high (only recombinants are produced). As mentioned by John Nash in an earlier response, small inserts give light blue colonies when a pUC-based vector is employed. Chris T. Amemiya Lawrence Livermore National Laboratory fish@amoeba.llnl.gov
msalminen@nphi.fi (03/16/91)
In article <1991Mar12.155321.1@csc.fi>, pollanen@csc.fi writes: > Hello > > Can anyone help me with m13 cloning I have some problems > with ligation a PCR product into m13 vector (m13mp18) > The size of fragments is varying from about 100bp to > 300bp size in lenght. I have opened the m13 vector > by pstI /HindIII double digestion and also I have > digested these PCR products same way because we have > planned primers so that PCR is (probably) producing > pstI and HindIII sites to the ends or near of the ends > of these fragments.... If anyone has tried to introduce > DNA fragments into m13 vector please let me know of > the problems and how did you solve those problems... > Especially talking about PCR fragment cloning into > m13 and also into expression vectors too... > > Sincerely yours Raimo Pollanen (M.S.) > > Pollanen@CSC.FI Hi Raimo! You could try two things: a) after PCR add 5-10 u Klenow. This repairs the ragged ends sometimes left by Taq. Then precipitate and digest. Precipitate again and clone. If you do a Kinase reaction the yield of inserts is better. Check the size of pcr-product before cloning by gel-electrophoresis. b) Do the fill-in with Klenow and precipitate. Blunt-end clone in suitable site such as Sal1. Again, kinase gives better yield. If you need more details, feel free to contact me at msalminen@finnphi (bitnet) or msalminen@nphi.fi (Internet) or nphi02::msalminen (Decnet) Mika Salminen KTL, HIV-Unit.
suttle@mbcf.stjude.org (03/16/91)
In article <1991Mar12.155321.1@csc.fi>, pollanen@csc.fi writes: > .... If anyone has tried to introduce (PCR) > DNA fragments into m13 vector please let me know of > the problems and how did you solve those problems... > Especially talking about PCR fragment cloning into > m13 and also into expression vectors too. I clone PCR fragments into M13 vectors all the time, by pretty much the same method you are trying to use. I design my primers so that a restriction site is included near each end. I can think of some problems that may be causing your cloning difficulties: 1. The primers should be designed so that the restriction cleavage site is near the center of @20mer. If you are hoping that the enzyme will cleave off the very last nucleotide or two of the PCR product, you may have trouble. A method was described in Nucleic Acids Research 18:6156 (1990) for "Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites" In this article, Jung et al. showed that if the PCR product is generated with 5'-Phosphorylated primers and then concatemerized with T4 DNA ligase, it is more readily digested at the terminal restriction sites. I have not had a reason to try this, but it sounds reasonable. 2. The M13 DNA may not be completely digested with both PstI and HindIII. There was a BRL Focus article about double digestions of the multiple cloning site (Focus 8:3 p. 9, 1986) in which they investigated the importance of using the restriction enzymes in particular order. They found that HindIII should be used before PstI and not the reverse. Also, it would be difficult to use them simultaneously because their sites are so close together. 3. You can verify that your M13 DNA is cut by both restriction enzymes by a simple test described in FMC Resolutions newsletter vol.2 no. 3. It basically involves taking aliquots of your DNA after both the first and second restriction digestion and digesting them together with an enzyme which cuts outside of the polylinker - like MstI. This produces two pieces of DNA which differ in length by the distance between the first two sites (like PstI and HindIII). Separation on 4% NuSieve agarose makes it possible to distinguish the two bands, which may be as few as 10 bp apart. I have used this protocol many times and would be glad to fax you a copy, if you need it. If none of these ideas solves your problem, you could directly sequence your PCR product, but that involves a whole new set of problems!!! Good luck! -- Barbara Bugg (also M.S.) St. Jude Children's Research Hospital Memphis, TN at SUTTLE@STJUDE.ORG