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GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
5. A 2% solution of LMP agarase in PBS is melted and kept at 50°C.
6. An equal volume ( 1 ml) of cell suspension and agarase is mixed well at
room temperature and immediately dispensed into the block formers.
7. Allow 20 min for the agarase to solidify; then push blocks through
molds using the sterile disposable plastic loops ( usually used to streak
out bacteria) directly into proteinase buffer. Add proteinase Kat 2 mg/
ml.
8. Keepblocks in proteinase K buffer 2-3 days at 50°C. Up to 100 blocks
can be placed in 50 ml proteinase buffer in a Falcon tube.
9. After proteinase K digestion, blocks can be stored in this buffer or in 0.5
M EDTA at 4°C.
10. Alternatively, continue the procedure by rinsing the blocks several
times with sterile TE buffer.
11. Put blocks in a Falcon tube containing TE plus 0.04 mg/ml PMSF to
inactivate remaining proteinase K.
Note: Caution! PMSF is poisonous! Incubate 2 x 30 min at 50°C. Replace 1 x
TE and PMSF after the first incubation.
12. Rinse blocks afterwards with several changes of TE at room temperature; return blocks to a clean tube; use directly for enzyme digestion or
store blocks in 0.5 M EDTA, (pH 8.0) at 4°C.
13. Afterstorage in EDTA, rinse block 2 x 30 min in TE at room temperature.
Subprotocol 2
Preparation of Size Markers
Materials
Lambda concatemers
Time required
Day 1: 1 h
Day 3: ready for use
1. Suspend clean concatemeric lambda DNA (Boehringer MA) in TE buffer
at 4 ).lg per 40 ).ll.
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
5. A 2% solution of LMP agarase in PBS is melted and kept at 50°C.
6. An equal volume ( 1 ml) of cell suspension and agarase is mixed well at
room temperature and immediately dispensed into the block formers.
7. Allow 20 min for the agarase to solidify; then push blocks through
molds using the sterile disposable plastic loops ( usually used to streak
out bacteria) directly into proteinase buffer. Add proteinase Kat 2 mg/
ml.
8. Keepblocks in proteinase K buffer 2-3 days at 50°C. Up to 100 blocks
can be placed in 50 ml proteinase buffer in a Falcon tube.
9. After proteinase K digestion, blocks can be stored in this buffer or in 0.5
M EDTA at 4°C.
10. Alternatively, continue the procedure by rinsing the blocks several
times with sterile TE buffer.
11. Put blocks in a Falcon tube containing TE plus 0.04 mg/ml PMSF to
inactivate remaining proteinase K.
Note: Caution! PMSF is poisonous! Incubate 2 x 30 min at 50°C. Replace 1 x
TE and PMSF after the first incubation.
12. Rinse blocks afterwards with several changes of TE at room temperature; return blocks to a clean tube; use directly for enzyme digestion or
store blocks in 0.5 M EDTA, (pH 8.0) at 4°C.
13. Afterstorage in EDTA, rinse block 2 x 30 min in TE at room temperature.
Subprotocol 2
Preparation of Size Markers
Materials
Lambda concatemers
Time required
Day 1: 1 h
Day 3: ready for use
1. Suspend clean concatemeric lambda DNA (Boehringer MA) in TE buffer
at 4 ).lg per 40 ).ll.