How to clone a 20 bp fragment?

William K. Pierce natldiagnos at mindspring.com
Thu Mar 20 05:00:58 EST 1997


Karin wrote:

Subject: 
            How to clone a 20 bp fragment?
       Date: 
            Thu, 20 Mar 1997 09:09:34 GMT
      From: 
            Karin.vdLee at FYS.rulimburg.nl (K van der Lee)
Organization: 
            Rijksuniversiteit Limburg
 Newsgroups: 
            bionet.molbio.methds-reagnts


Hi,

I am trying to clone a 20-25 bp fragment into a 6.1 kb vector. They both
contain 5' overhang ends (XhoI and BamHI). Although I tried many things, 
nothing worked out until now. Any suggestions???

Thanks,

Karin.


KARIN,
 
YOU DO NOT SAY WHAT IT IS THAT IS "NOT WORKING"....IT WOULD BE USEFUL TO
KNOW MORE BEFORE WE TROUBLE-SHOOT. HOWEVER, LET ME OFFER THE FOLLOWING
POSSIBILITY:

INFREQUENTLY, BUT JUST OFTEN ENOUGH TO BE A PROBLEM, WHEN CLONING A
SMALL FRAGMENT THE INSERTION MAY NOT INACTIVATE THE BETA-GALACTOSIDASE
(ASSUMING THIS IS YOUR "ASSAY").

FOR EXAMPLE: LIGATION OF A 24-BASE PAIR INSERT WILL ADD EXACTLY EIGHT
CODONS. IT IS POSSIBLE TO GET ALL BLUE COLONIES AND STILL HAVE LIGATED
THE 24 BASE PAIR INSERT INTO THE VECTOR IF: 
1. THE DNA INSERT LIGATED INTO THE VECTOR "IN REGISTER" WITHIN THE READ
FRAME OF THE BETA-GALACTOSIDASE
2. EACH OF THE EIGHT CODONS ACTUALLY CODES FOR AN AMINOACID 
3. THOSE EIGHT AMINO ACIDS DO NOT SIGNIFICANTLY ALTER THE ACTIVITY OF
THE BETA-GALACTOSIDASE.

IF THE ABOVE THREE CONDITIONS ARE SATISFIED, THEN YOU WILL GET "ALL BLUE
COLONIES", AND YOU WOULD (INACCURATELY) CLAIM THAT YOUR LIGATION DID NOT
WORK.

A WAY TO AVOID THIS PROBLEM IS TO DEPHOSPHORYLATE THE VECTOR AND RUN
APPROPRIATE CONTROLS: 
TO BE CERTAIN EACH OF THE ENZYMES YOU HAVE USED TO LINEARIZE THE VECTOR
ACTUALLY CUT THE VECTOR
TO BE CERTAIN THE LIGASE IS WORKING
TO BE CERTAIN THE CELLS ARE COMPETENT
ETC., ETC.

SELECT SOME OF YOUR TRANSFORMANTS TO SEE IF INDEED THAY HAVE
ACCEPTED THE INSERT.....WITH APPROPEIATE CONTROLS AND DEPHOSPHORYLATED
VECTOR, ETC. YOU CAN BE RELATIVELY CERTAIN THAT  A BLUE COLONY CONTAINS
THE INSERT, IN-REGISTER, AND THE INSERT SIMPLY DID NOT INACTIVATE THE
BETA-GALACTOSIDASE.

I'VE HAD IT HAPPEN TO ME......THANK GOODNESS I'D CIP'D MY
VECTOR.......SURE ENOUGH EVERY BLUE COLONY I CHECKED HAD ACCEPTED A 33
BASE PAIR INSERT, IN REGISTER, ETC.


HANK DAUM, III
NATIONAL DIAGNOSTICS, INC.



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