single cell PCR

Karthik Aghoram kaghoram at unity.ncsu.edu
Thu Jun 14 12:35:35 EST 2001


It is amazing.  But, I did my grad work in a lab where we routinely
dissected single guard cells from frozen-dried tissues of various plants. 
The cells were then lifted one-by-one and placed in reaction mixtures to 
measure sucrose, ABA, malic acid, enzyme activities .... you name it.

We tried RT-PCR once, with no success.  But recently many protocols have 
been developed for single-cell gene exp'n analysis.  Pharmacia even sells 
a kit for this purpose. 

I apologize if you already knew this stuff and were just kidding :-)!  
Couldn't resist sharing this stuff here!

Karthik


R. Jayakumar (jakku at mrna.tn.nic.in) wrote:

: hi...
:   BOy! that is a rare talent , I mean putting a single CELL into a PCR tube.
: How do you do it?  The best I could do was putting a single colony.. but a
: single cell.. . that is something.  Moreover,  how do you incubate it at
: 650C.  that must be damn pretty hot.... Do you use a silica crucible for
: that.......???
:      nice technique..:-)))
: jakku
: 
: ----- Original Message -----
: From: "water" <s6311190 at ms22.hinet.net>
: To: <methods at hgmp.mrc.ac.uk>
: Sent: Thursday, June 14, 2001 9:26 PM
: Subject: single cell PCR
: 
: 
: > 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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