PCR cloning - a protocol

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Fri Mar 11 15:31:42 EST 1994


To All:  I have received a number of requests about details concerning
our method of TA cloning.  I enclose some of the questions and a
detailed protocol that I use for subcloning.  We use this protocol to
make libraries of T cell receptor gene products from small amounts of
tissue.  Good Luck.





> However, when I 
> tried it (making my own vector using the protocol you mentioned) 
>  I met with failure again.  You seem to have working the exact technique 
> which I would like to use, so I'd like to pose a few questions.  
> 1)  In your opinion, should there be any preference between using NEB HincII 
> or NEB SmaI? 
> 2)  How fresh must the vector and PCR product be? (I've heard 
> tales of the single A and T bases falling off).   

In vitrogen says the PCR product should be used fresh.  I have not
tested this myself. I always do it fresh.

3)  What ratio of insert:vector works best for you and how do you go
about 
> figuring that out?

I don't figure it out.  I just guess (see the enclosed protocol)

>Brian Zeiler 
> Dept. of Microbiology and Molecular Genetics, UCLA
> brianz at lifesci.microbio.ucla.edu


 ***Enclosure of Protocol begins here***  

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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