hoechst dna stain

From:Patsy.Ruegg@UCHSC.edu

 

-----Original Message-----
From: rueggp
To: patsy.ruegg@uchsc.edu
Sent: 4/2/01 7:44 PM
Subject: dna stain

Reference: Cesar La Barca and Kenneth Paigen
A Simple~ Rapid~ and Sensitive DNA Assay Procedure. Analytical Biochern
102, 344-352.
I. Stock Solutions
A. Hoechst Dye Stock Concentrate
0.5 mg/mi Hoechst dye in water.
Store protected from light, at 4C, for up to 6 months. Working
Solution: 4 ul in 10 mi 4.6 M NaCI-PO4 buffer. Make up fresh before each
use.
B. DNA STANDARD STOCK
5 mg calf thymus DNA in 10 mi 5 mM NaOH
Allow to stir overnight at 4C, pipet to further dissolve. Aliquot into
10 mi sterile tubes (200 ul) and store at -20C.
C. 4.6 M NaCI-PO4 buffer
1.14 g/L NaH2PO4 (anhydrous)
5.753 g/L Na2HPO4 (anhydrous) 268.6 g/L NaCI
Adjust pH to 7.4
D. 1M NaOH
E. 1M HCI+200mM PO4
500 mi X (50 mM NaH2PO4)
(150 mM Na2HPO4) Adjust pH to 7.4
Discard 41.67 mi of buffer, adjust volume to 500 mls with 12 M HCI.
VERY IMPORTANT
It is now necessary to ensure that the NaOH and
1M HCI+200mM PO4 are of equal molarities. To a 50 ml tube, add 5 mis of
each, and check the pH. If other than pH 7.4 adjust concentrations until
result of the addition is 7.4.
F. 0.25 M NaCI

 ." ".;."'..""c."""'",..,,
II. CELL PREPARATION
A. Aliquot 100 ul sample ~to 1.5 mI tubes. Add 200 ul
1 M NaOH. Vortex well and freeze at -20C. Incubate 1 hr at room
temperature, or overnight at 4C.
B. Add 200 ullM HCI+200mM PO4. Sample is now ready.
III. PREPARATION OF DNA STANDARD CURVE A. DNA SOLUTION A
Remove 1 tube of DNA stock solution from -20C freezer. Add 1.9 mIIM
HCI+200mM PO4 and 1.9 mI NaOH. B. DNA SOLUTION B
Tol.6 mI 0.25 M NaCI, add 400 ul DNA SOLUTION A.
IV .DNA ST ANDARD CURVE
Add directly to microflour 96 well plate the following amounts:
DNA final concentration 0.25 M NaCI DNA A ~NA B
3.2 ug/mI 93 ul 32 ul
1.6 ug/mI 109 ul 16 ul 0.8 ug/mI 117 ul 8 ul
0.4 ug/mI 105 ul 20 ul 0.2 ug/mI 115 ul 10 ul 0.1 ug/ml 120 ul 5 ul 0.0
ug/mI 125 ul
You must load plate in dupicate only, starting from highest
concentration and going to lowest, in 125 ul volumes.
A. Add 125 ul of sample into remaining wells, in duplicate.
B. Add 125 ul Hoechst dye (of working concentration) to each well.
Incubate in light protected environment, for at least 20, but less than
60 minutes
C. Read on Dynatech Microflour plate reader for relative
flourescence units. Estimate the DNA content of samples from standard
curve.





<< Previous Message | Next Message >>