Experiment
log
ISH-chemicals and solutions
Alcohols
TRIS-HCl
DTT (DiThioTreitol)
NaAc (sodium acetate)
Salmon for hybridisation
blocking
Hybridisation
cocktail
/Formamide
/20 x SSC
/Denhardt´s
solution 100x
/N-Lauroylsarcosine
/NaPO4 buffer
Alcohols
Methanol (Conc.)
Store at 4°
Ethanol (20%)
Dilute with autoclaved ddH2O
Store at 4°
TRIS-HCl
0.1M (pH: 8.0)
(TRIS Mw121; HCl Mw 36.5)
6.05g TRIS löses i 400ml autoklaverat ddH2O
justera till pH 8.0 med HCl (37%); buffrar bra mellan 8-9, dock
sämre kring8 à försiktighet!!
addera ddH2O till slutvolym 500ml
Store at 4o
OBS! TRIS-HCl temp. beroende pH, +1oCà-0.028 pH-enheter;
pH 8.0 kalibrerat vid +13-15oC
DTT (DiThioTreitol)
DTT (DiThioTreitol) 0.5M / 5M
(DTT Mw-154.24)
5 g DTT / 3240µl = 10M
5g lyophiliserad DTT löses i 3240µm 0.01M NaAc-buffert(pH
5.2)+à 5M DTT
vortexa ca 10min (-> 6500-6600 µl total volume; volume
effect!?)
aliquotera till autoklaverade eppendorfrör (600µl)
från 5M DTT görs 0.5M DTT, späd med 0.01M NaAc-buffert
enl. ovan
Store at -20o (DTT very unstable, thaw immediately before use!)
NaAc (sodium
acetate)
NaAc 0.01M (pH 5.2) (NaAcetat Mw-82.03)
Gör 500ml stock 1M NaAc-buffert:
41g NaAc (anhydr.) löses i 400ml ddH2O
justera pH till 5.2 med glacial acetic acid (isättika)
addera ddH2O till slutvolym 500ml
späd stamlösning 100x à 0.01M NaAc-buffert
Store at 4oC
Salmon
for hybridisation blocking
Lax (salmon testis DNA type III, Na-salt)
dilute Lax à(10 mg/ml) in ddH2O (lämpligen 100mg
lax till 10ml ddH2O, i 15ml falconrör)
sonikera (histologen) på full speed 3x15s eller till det
är helt löst (OBS! Hörselskydd, tvätta av sonikator)
aliquotera till autoklaverade eppendorfrör (1000µl)
Store at -20o
Hybridisation
cocktail
-
Use gloves and autoclaved equipment
To make approximately 200 ml:
1. 100 ml Formamide
2. 40 ml 20 x SSC
3. 2 ml Denhardt´s
100x
4. 10 ml 20% N-Lauroylsarcosine
(Sarcosyl)
5. 20 ml 0.2 M NaPO4 buffer
(pH 7.0)
6. 20 g Dextransulphate
-
Thaw solutions at room temperature
-
Dissolve while stirring overnight in a light secure (Al-foil) E-flask
-
Aliquot to 15ml falcon tubes, wrap in Al-foil
Store at -20°C until use
Thaw at 42°C before use and filter if not recently prepared
(0.45µm filter)
Formamide
avjoniseras tills pH<5.6 (joniserat- pH>10); svårt få
ner till 5.6, OK med < 8-9 (6.8)
use Mol Biol grade mixed bed resin (20-50g mesh) 50g/500 ml Formamide
omröres i rumstemp overnight (autoklavera omrörare)
filtrera genom filterpapper (filterpapper #3)
aliquotera till 50ml falconrör
Store at -20o inslagna i Al-folie
20 x SSC
Lös 175.3g NaCl och 88.2g NaCitrat-2-hydrat i 800 ml ddH2O
Adjust pH to 7.0 (NaOH/HCl)
Adjust volume to 1000 ml using ddH2O
Store in RT
Denhardt´s
solution 100x
to make 100 ml: (görs lämpligen i 2x50ml falconrör)
2g Ficoll
2g BSA
2g Polyvinylpyrrolidone
löses i ddH2O till 100ml
filtersterilisera (0.22µm filter)
Store at -20oC i 15/50ml falconrör
N-Lauroylsarcosine
Dissolve 20g of N-Lauroylsarcosine in
80ml autoclaved ddH2O
Add ddH2O to final volume of 100ml
Store at room temperature, cover with Al-foil
0.2M NaPO4
buffer (pH 7.0)
to make 500ml:
17.8g Na2HPO4 x 2 H2O (mw-177.99)
13.8g NaH2PO4 x 1 H2O (mw-137.99)
löses i 400ml ddH2O
justera pH till 7.0 (NaOH)
addera till slutvolym 500ml
Store at 4°C
Suggestions and Comments