Ligation techniques

Patrick Helmer James Falckh phjf at ariel.ucs.unimelb.EDU.AU
Thu Nov 5 20:19:13 EST 1992



Hi Bio-netters,
	I am looking for references on how to perform ligation of cDNA and
single strand oligos.  I am not a biochemist but a pharmacologist muddling my 
way throught molecular biology techniques and utilise RT/PCR to 
investigate changes in the expression of receptor types; and subtypes.  I
have seen several different ways to quantitate PCR products and have designed
my own standard that I wish to construct.

	My approach is two fold : 
	1)  To join several oligos together with some "junk" in between, that
will be inserted in Bluescript with T3 and T7 promoters; the idea is similar
to that of Wang et al (Proc. Natl. Acad. Sci USA, Vol 86:9717-9721). This
will allow me to create cRNA that can be included in the initial RT reaction
and adjust for efficiency;
	2)  To 'cut and paste' several sections from different sequenced
segments of PCR products that I presume will require the use of several
different restriction enzymes and linkers/adapters.

	From my reading both methods seem feasible; if not practical.

	Anyone with information (or constructive criticism) on how to go
about the construction of either two methods, or where to get the info from
journals would be greatly appreciated.



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+                               |                                +       
+    Patrick HJ Falckh PhD      |                                + 
+    Dept of Pharmacology       |   Telephone : 61-03-344 6992   +  
+    University of Melbourne    |    Fax No   : 61-03-347 1453   + 
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