salger at wap18.zi.biologie.uni-muenchen.de
Sun Feb 19 16:44:45 EST 1995
HHOFF at CRCVMS.UNL.EDU wrote:
: We're interested in the method mentioned by a netter recently using
: Taq polymerase and dTTP to put a T overhang on your favorite vector
: (to be used in home-made TA cloning). Unfortunately, I had already
: the message by the time I mentioned it to my labmate, who needs more
: We'd greatly appreciate a more detailed protocol.
: Thanks in advance, Heidi
here's a reference that is worth reading if you want to clone PCR products
and the requested protocol by James F. George (posted on Sept 29 1994).
Klaus Salger phone : ++49 (0)89 5902 -502
Zoologisches Institut FAX : -450
AG MacWilliams e-mail: salger at zi.biologie.uni-muenchen.de
Reference about cloning PCR products: Chuang et al. TIG 11, p.7, January 95
And here comes James George's protocoll:
txpljfg at UABCVSR.CVSR.UAB.EDU wrote:
: TA Subcloning of PCR Products
: Tvector.doc James F. George, Ph.D.
: This procedure is adapted from D. Marchuk, M. Drumm, A.
: Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154.
: CONSTRUCTION OF T-VECTOR
: 1. suspend 10ug pUC 19 in:
: 4.0ul 10X reaction buffer (we use Bo. Mann. buffer A)
: 2.0ul (20U) Sma I
: xx.Xul dwater to a total vol. of 40ul
: Incubate at 30 (not 37) degrees for 1 hour.
: This is easier if done in a 0.4ml tube in a thermal
: 2. Heat to 70 degrees for 15 min. to kill the enzyme
: 3. Bring to 100ul w/ water (add 60ul).
: 4. Extract w/ phenol, phenol/chloroform and then
: 5. add 9ul 3M sodium acetate.
: 6. ppt. in ETOH, wash with 70% ETOH (be careful with the
: 7. Dry in spin vac at room temp (do not use heater!).
: *********************T-TAILING THE VECTOR******************
: At this point, it is assumed that there has been 80%
: recovery of the cut plasmid DNA.
: 1. Resuspend the plasmid DNA in 63ul water (conc approx.
: 2. To the resuspended plasmid add:
: 10ul 10X PCR buffer (standard cetus stuff,
: no MgCl)
: 20ul 10mM dTTP [2mM final]
: 6ul 25mM MgCl2 [1.5mM final]
: 1ul Taq polymerase (Cetus amplitaq 5U/ul)
: 100ul total volume.
: 3. Incubate for 3 hours at 70 degrees C.
: 4. Extract with Phenol, Phenol/chloroform, chloroform.
: 5. Extract twice with ether (so I'm paranoid!)
: 6 add 75ul 2M **ammonium** acetate (assuming 75ul recovery
: from extractions).
: 7 Add 150ul isopropanol. Spin 20mins in microfuge at full
: speed at 4 degrees.
: 8. Wash with 70% ETOH
: 9 Dry pellet in spin vac and store at -20 degrees until
: TREATMENT THE PCR PRODUCTS
: " If you can see it, you can clone it".
: 1. Add an equal volume of chloroform (*NO* IAA) to the PCR
: reaction and spin 1-2 minutes in microfuge at RT.
: 2. Remove the oil which is now on the ****BOTTOM***.
: 3. Spin again for two minutes and remove the last little
: bit of oil from the bottom. You will know when you have
: gotten it all when you see the interface in the pipette
: tip. It is important that all the oil be removed
: otherwise subsequent procedures will be very difficult.
: 4. Add 100ul 4M ammonium acetate, vortex, and then add
: 200ul isopropanol.
: 5. Centrifuge 20min at 4 degrees, wash in 70% ETOH.
: 6. Dry in speed vac.
: 7. Resuspend the DNA in 8-10ul TE, add loading buffer and
: load onto a 4% Nusieve (TAE) agarose gel. Run until the
: desired band is well separated. The more DNA in the
: band, the easier it is to subclone.
: 8. Cut out the band. Minimize the exposure of the gel (and
: you!) to short wave UV
: LIGATION OF PCR PRODUCTS TO T-VECTOR
: 1. Heat the gel containing the PCR fragment to 65C for 10
: minutes, place in a 37C water bath or block and add to a
: separate tube (also at 37C):
: 10 ul gel
: 4ul 5X ligase buffer (commercial buffer
: that comes with BRL T4 ligase)
: 4ul water
: 1ul vector (25-50ng)
: 1ul ligase
: Incubate at 12C overnight.
: 2. Heat the mixture to 68 degrees for 5 minutes and add
: 100ul water.
: 3. Extract with phenol, phenol/chloroform, and chloroform.
: These steps are to remove the agarose.
: 4. Add 10ul 3M NaAcetate and precipitate with ethanol.
: 5. Wash the pellet in 70% ETOH, dry in the speed-vac.
: Resuspend in 5ul of water just prior to transformation.
: *Transformation - We usually use electroporation into
: XL-1 blue cells. You need cells that can achieve
: at least 1 x 10^7 transformants per ug of DNA if a
: CaCl based protocol is used.
: *Storage: The T-vector should be stored at -20C at all
: times. When stored in dry form, the T-overhangs
: will last longer (I don't know how long yet). In
: solution, it lasts at least a couple of weeks at -
: *Enzymes - The batch of SmaI that is used is
: particularly critical. Some are contaminated with
: an endonuclease that removes a few bases from the
: cloning site. The batch of smaI should be checked
: before it is used to cut vector for cloning
: purposes. If bluescript is used, EcoRV can be
: substituted for sma I.
: Subcloning PCR products James F. George, Ph.D.
: James F. George, Ph.D. "Back off man, I'm a scientist"
: Department of Surgery --Bill Murray
: University of Alabama at Birmingham
: 205-934-4261 voice
: txpljfg at uabcvsr.cvsr.uab.edu
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