single cell PCR

Karthik Aghoram kaghoram at unity.ncsu.edu
Thu Jun 14 11:06:58 EST 2001


Did you try to amplify an abundant gene from a single cell as a positive
control (something that has worked for somebody somewhere)?  If that
didn't work either, then your cells are probably not lysing; so you have 
no template to begin with.  Any takers for this theory?


water (s6311190 at ms22.hinet.net) wrote:
: Hi,
: I am trying to run single cell PCR. Now I encounter a problem. After the
: second PCR, there was no product.
: 
: PCR condition:
: First, I put a cell into a 0.2 ml PCR tube with 5 ul alkaline lysis
: buffer (50 mM DTT, 200 mM KOH). Then incubate it at 650C for 5 min.
: After incubation, 5 ul neutralization buffer (900 mM Tris-HCl, 300 mM
: KCL, 200 mM HCl) was added to the mixture. Then the mixture was brought
: to a final volume of 50 ul (10 mM Tris-HCl, 50mM KCl, 1.5mM MgCl2, 0.25
: mM each of the four dNTPs, 0.5U/ul Taq DNA polymerase, 1uM each of
: primers).
: 
: Pre-PCR  94oC, 5 min
: 
: PCR     94oC, 1 min
:             55oC, 1 min
:             72oC, 1 min
:            33 cycle
: Post-PCR  72oC, 5 min
: 
: Then 2 ul fist PCR product was used as template for same PCR. The
: condition was as above including primers.
: 
: The PCR condition described above works fine with genomic DNA.
: Could anyone make a suggestion to me.
: 
: Thank you for help!
: 
: Sincerely, Chang Ya-Hui
: 2001/6/14
: 
: 
: ---

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Karthik Aghoram				Postdoctoral Associate
					Dept of Crop Science
e-mail: kaghoram at unity.ncsu.edu		North Carolina St University
Phone (W): 919-515-2705			Raleigh, NC 27695-7620

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