Thermo Scientific Shake 'n Stack 6244, Operating And Maintenance Manual

Page: 56 / 92

© Thermo
Scientific
, May 2003. Issue 7 3
CHAPTER 2
HYBRIDISATION GUIDE
NUCLEIC ACID TRANSFER PROCEDURES
Dot/Slot Blotting
1. DNA samples, prepared from cell lysate, must first be denatured to form single-
stranded DNA. Heat to 100
°
C and incubate for 5 minutes.
2. Chill DNA samples on ice for 5 minutes.
3. Cut two pieces of Whatman 3MM

filter paper and one sheet of membrane to the
size of the Dot blotting unit. Pre-wet the membrane and filter paper in 6 x SSPE
(SSC), then place in the unit and secure the lid. If samples are to be spotted on
manually, pre-wet the membrane in 6 x SSPE (SSC) and place on dry filter paper.
4. Pipette samples into the wells for binding of the DNA to the membrane and follow
manufacturer’s instructions to operate apparatus correctly.
If samples are spotted manually, apply 0.5-1.0
µ
l aliquots and allow to dry between
applications to prevent excessive spreading.
5. Dismantle the apparatus and remove the membrane.
6. Place the membrane on a sheet of Whatman 3MM

filter paper saturated with
denaturing solution for 1 minute.
7. Transfer the membrane to a sheet of filter paper saturated with neutralising
solution for one minute.
8. Place the membrane on dry filter paper and allow to air dry.
9. Fix the DNA by baking the membrane for 1 hour at 80
°
C, or by UV cross-linking.
UV cross-linking is the most efficient method of fixing the DNA to Hybridisation
membranes and is particularly useful if reprobing is to be carried out.
10. After fixing the DNA the membrane is ready for Hybridisation. Membranes not used
immediately may be stored between sheets of Whatman 3MM

in sealed plastic bags
at 4
°
C.