PCR of Lambda Inserts Question

fzhedric at hamlet.ucdavis.edu fzhedric at hamlet.ucdavis.edu
Fri Jul 10 16:52:56 EST 1992


In article <3553 at fcs280s.ncifcrf.gov> pnh at fcsparc6.ncifcrf.gov (Paul N Hengen) writes:
>Mike: Why not post a summary of responses to the net? Other people
>  (including me) from all over the world follow this newsgroup for
>  the same reason you do...Thanks.
>
>Paul N. Hengen
>National Cancer Institute
>Frederick Cancer Research and Development Center
>Frederick, Maryland 21702-1201 USA
>pnh at ncifcrf.gov

Hello,
	Well, I received a lot of suggestions and they generally broke down 
as follows:
	1) Criteria for taq polymerase funtion
		Needs Mg for activity so include ~1mM to .1mM in solution
	2) Lambda DNA must be released
		Do either a freeze/thaw before amplification or
		boil the sample (in water bath) 5 min prior to amplification
		and addition of taq polymerase.  I wonder why this must be
		done if the reaction is cycled through 94 C.  I would
		think that at this temp. most phages would fall apart.  Maybe
		someone could clarify this for me.
	3) Be careful not to add too much DNA template
		Someone recommended making a dilution series of my phage when
		I amplify (1:10 to 1:1000 in 1:10 increments)

	Philip Moos sent me a protocol he has had success with and here it is:
			(thanks Philip)
                       "32 ul ddH2O
                        2 ul high titer lysate
                        5 ul 10x PCR buffer (with 1.5 mM MgCl2)
                        5 ul 2mM dNTP's
                        2 ul each primer (10pmole)

                boil 5 min.

                        add 1.25 Units of Taq


                cycle as follows:

                        94 C  for 30 sec.
                        55 C  for 30 sec.
                        72 C  for 1-3 min. (depending on insert size)
                                (35 cycles)
                        finish with  72 C for 5 min.

        I have a hard time believing this could be run without Mg -- I've
done tests for Mg concentration -- maybe the buffer the virus was in had
high amounts of Mg or something.  I also have not used Tween -- DMSO and
PEG are supposed to increase specificity -- I have not required those
either (using this protocol)."   -Philip Moos

					Thanks to all the submitters
					and good luck,
					Mike Oda
					fzhedric at hamlet.ucdavis.edu

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My boss doesn't known what I am doing... |  Biochem Grad. Student
So please don't tell him.		 |  fzhedric at hamlet.ucdavis.edu
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