TA Cloning

[Klaus Salger]

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
: deleted
: the message by the time I mentioned it to my labmate, who needs more
: details.
: We'd greatly appreciate a more detailed protocol.
: Thanks in advance, Heidi

Dear 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).

Cheers
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany


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 
: cycler. 
:  
: 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 
: chloroform. 
: 5. add 9ul 3M sodium acetate. 
: 6. ppt. in ETOH, wash with 70% ETOH (be careful with the 
: pellet!). 
: 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. 
: 130ng/ul) 
: 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 
: use.


: 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 -
: 20C.  
:  
: *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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