30 Microdissection of Chromosomes and Reverse FISH
391
- Dextran sulfate (Serva, #18705)
- Tween 20 (Sigma, #P-1379)
- Human Cot-l DNA (Life Technologies, #15279-011)
- NaOH
- DAPI (4',6-Diamino-2-Phenylindole, Sigma, #D 9542)
- Vectashield mounting medium (Vector laboratories, #H-1000)
- PBS (Life Technologies, #14190-094)
- Fixogum (Marabu, #2901 10 000)
Note: In order to reduce the risk of DNA and nuclease contamination,
it is essential to wear gloves when handling any chemicals and liquids.
All chemicals should be restricted for use in microdissection experiments only.
Any chromosome preparations harvested following routine protocols can
be used. Cell suspensions stored in Carnoy's ftxative at -20°C for several
years have been successfully used. However, treatment with intercalating
substances (e.g. ethidium bromide) should be avoided, because they seem
to reduce the PCR efftciency signiftcantly.
Procedure
Preparation of metaphase spreads
In order to obtain adequate spreading of metaphases, the coverslips have
to be cleaned. For this purpose they are soaked in a 2% SDS solution, at
least overnight. Prior to use, they are rinsed thoroughly under running tap
water and thereafter twice in sterile double distilled H 2 0 (dd H 2 0). Dissection of the chromosomes is preferably carried out on freshly spread
metaphases. They become harder with age and excision of deftned chromosomal fragments becomes inexact. Moreover, excised fragments become fragile and tend to break into several small pieces, which makes
them more difftcult to collect.
The cell suspension is dropped onto the wet coverslips and air dried.
Chromosome
preparation
391
- Dextran sulfate (Serva, #18705)
- Tween 20 (Sigma, #P-1379)
- Human Cot-l DNA (Life Technologies, #15279-011)
- NaOH
- DAPI (4',6-Diamino-2-Phenylindole, Sigma, #D 9542)
- Vectashield mounting medium (Vector laboratories, #H-1000)
- PBS (Life Technologies, #14190-094)
- Fixogum (Marabu, #2901 10 000)
Note: In order to reduce the risk of DNA and nuclease contamination,
it is essential to wear gloves when handling any chemicals and liquids.
All chemicals should be restricted for use in microdissection experiments only.
Any chromosome preparations harvested following routine protocols can
be used. Cell suspensions stored in Carnoy's ftxative at -20°C for several
years have been successfully used. However, treatment with intercalating
substances (e.g. ethidium bromide) should be avoided, because they seem
to reduce the PCR efftciency signiftcantly.
Procedure
Preparation of metaphase spreads
In order to obtain adequate spreading of metaphases, the coverslips have
to be cleaned. For this purpose they are soaked in a 2% SDS solution, at
least overnight. Prior to use, they are rinsed thoroughly under running tap
water and thereafter twice in sterile double distilled H 2 0 (dd H 2 0). Dissection of the chromosomes is preferably carried out on freshly spread
metaphases. They become harder with age and excision of deftned chromosomal fragments becomes inexact. Moreover, excised fragments become fragile and tend to break into several small pieces, which makes
them more difftcult to collect.
The cell suspension is dropped onto the wet coverslips and air dried.
Chromosome
preparation