Appendix D: Preparation of Solutions

Transcription

1 1. Solutions for DNA and RNA Labeling Appendix D: Preparation of Solutions 1. Solutions for DNA and RNA Labeling The reagents required for labeling depend on the labeling method chosen. For a complete list of reagents required for each labeling method, see the following sections of : For this labeling method... Reagents are listed in... Starting on page Random primed labeling of DNA Section 2.1.1, PCR labeling of DNA Section 2.2.1, Transcriptional labeling of RNA Section 2.3.1, Direct labeling of DNA or RNA Section 2.5.1, Solutions and Buffers for DNA/Southern Blotting and Hybridization Water Always use autoclaved, double distilled H 2 O for all Southern blotting/hybridization solutions and buffers. DNA gel running buffer 89 mm Tris-borate 2 mm EDTA Adjust ph to 8.3 (25 C). TE buffer 10 mm Tris-HCl 1 mm EDTA Adjust ph to 8.0 (25 C). DNA probe dilution buffer (for estimate of probe yield) TE buffer containing 50 µg/ml herring sperm DNA (included in the DIG-High Prime Labeling and Detection Starter Kit II) Depurination solution 0.25 M HCl Denaturation solution (for Southern transfer or colony/plaque hybridization) 0.5 M NaOH Neutralization solution 1 (for Southern transfer) 0.5 M Tris-HCl, ph

2 3. Solutions and Buffers for RNA/Northern Blotting and Hybridization Neutralization solution 2 (for colony/plaque hybridization) 1.0 M Tris-HCl, ph 7.4 DIG Easy Hyb hybridization buffer Option 1: Purchase ready-to-use, DNaseand RNase-free DIG Easy Hyb solution ). Use as supplied for prehybridization or add the appropriate amount of labeled DNA probe for hybridization. Option 2: Purchase DNase- and RNasefree DIG Easy Hyb granules as an individual reagent (Cat. No ) or as part of the DIG-High Prime Labeling and Detection Starter Kit II (Cat. No ). Add 64 ml sterile, double distilled water (in two aliquots) to 1 portion of granules. Stir the solution for 5 min at 37 C until granules dissolve. Use reconstituted DIG Easy Hyb without further additives for prehybridization or add the appropriate amount of labeled DNA probe for hybridization. SDS stock solution (Cat. No ) Dissolve 10% (w/v) sodium dodecyl sulfate in water. Filter through a membrane ( µm). Low stringency wash buffer High stringency wash buffer 0. (1:40 dilution of stock 20x SSC) Probe stripping solution (for DNA probes labeled with alkali-labile dutp) 0.2 M NaOH Membrane wash buffer (after probe stripping) 20x SSC (Cat. No ) 3 M NaCl 0.3 M sodium citrate Adjust ph to 7.0 (20 C) and autoclave. 3. Solutions and Buffers for RNA/Northern Blotting and Hybridization Water Always use autoclaved, DMDC- or DEPC-treated, double distilled H 2 O for all Northern blotting/hybridization solutions and buffers. DMDC-/DEPC-treated water is RNase-free. Preferred treatment: Dissolve 1% (e.g. 1 g per 100 ml) dimethyl-dicarbonate (DMDC) in a 50% ethanol/water mixture. Mix double distilled H 2 O 1:10 with the stock 1% DMDC solution (final concentration, 0.1% DMDC). Incubate for 30 min at room temperature, then autoclave the treated water. Note: You can also use DEPC (diethylpyrocarbonate) to treat the water. However, we prefer DMDC, since it is less toxic than DEPC. RNA probe dilution buffer (for estimate of probe yield) Mix DMDC- or DEPC-treated water, 20x SSC, and formaldehyde in the ratio 5:3:2. Note: Always treat water and 20x SSC with DMDC or DEPC before use (to destroy RNases). 206

3 3. Solutions and Buffers for RNA/Northern Blotting and Hybridization 10x MOPS buffer 200 mm morpholineopropanesulfonic acid (MOPS) 50 mm sodium acetate 20 mm EDTA Adjust ph to 7.0 with NaOH. Autoclave. Note: The solution will turn yellow after it is autoclaved. This will not affect its performance. Dilute 1:10 to make RNA gel running buffer. Deionization of formamide Add 50 g AG 501-X8 ion-exchange resin to 500 ml formamide. Stir slurry slowly for 30 min on a magnetic stirrer. Remove the resin by filtration and store the filtered, deionized formamide at 20 C. RNA loading buffer Just before use, prepare a solution containing the following: 100% formamide, deionized 250 µl 37% (w/v) formaldehyde 83 µl 10x MOPS buffer 50 µl 2.5% bromophenol blue 10 µl 100% glycerol, RNase-free 50 µl DMDC-/DEPC-treated H 2 O To make 500 µl total volume (requires approx. 57 µl) DIG Easy Hyb hybridization buffer Purchase DNase- and RNase-free DIG Easy Hyb (Cat. No ), ready-touse or DIG Easy Hyb granules as an individual reagent (Cat. No ) or as part of the DIG-High Prime Labeling and Detection Starter Kit II (Cat. No ). Just before use, add 64 ml sterile, DMDC-/DEPC-treated, double distilled water (in two aliquots) to 1 portion of granules. Stir the solution for 5 min at 37 C until granules dissolve. Use reconstituted DIG Easy Hyb without further additives for prehybridization or add the appropriate amount of labeled RNA probe for hybridization. Low stringency wash buffer High stringency wash buffer 0.1x SSC (1:200 dilution of stock 20x SSC; see Section 2 above for 20x SSC) Probe stripping solution (for RNA probes) 50% formamide 50 mm Tris-HCl, ph 7.5 5% SDS Membrane wash buffer (after probe stripping) 207

4 4. Required Solutions and Buffers for Detection 4. Required Solutions and Buffers for Detection Note: For detection of RNA hybrids on Northern blots, always use DMDC- or DEPC-treated water to make all detection reagents (see Section 3 above). Observe RNase-free conditions when making the reagents. Maleic Acid Buffer To make working Maleic Acid Buffer, dilute the stock buffer 1:10 with sterile, double distilled water. Washing Buffer Before use, shake the stock solution to resuspend the buffer components. To make working Washing Buffer, dilute the stock buffer 1:10 with sterile, double distilled water. Blocking Solution Purchase sterile, nuclease-free, stock solution (10x concentrated) as part of the DIG Wash and Block Set (Cat. No ), the DIG-High Prime Labeling and Detection Starter Kit II (Cat. No ), or the DIG Northern Starter Kit (Cat. No ). To make working (1%) Blocking Solution, dilute the stock solution 1:10 with working Maleic Acid Buffer just before use. Detection Buffer To make working Detection Buffer, dilute the stock buffer 1:10 with sterile, double distilled water. Chemiluminescent substrate solution Option 1: Purchase ready-to-use CDP- Star ) or as part of the DIG Northern Starter Kit (Cat. No ). Use as supplied. Option 2: Purchase ready-to-use CSPD as an individual reagent (Cat. No ) or as part of the DIG-High Prime Labeling and Detection Starter Kit II (Cat. No ). Use as supplied. Color substrate solution Option 1: Purchase NBT/BCIP stock solution ) or as part of the DIG-High Prime Labeling and Detection Starter Kit I (Cat. No ). Just before use, add 200 µl of the stock solution to 10 ml of Detection Buffer to make Color Substrate Solution. Option 2: Purchase NBT/BCIP tablets ). Just before use, add one tablet to 10 ml of sterile, double distilled water to make Color Substrate Solution. Option 3: Purchase red, green, and blue substrate tablets as part of the Multicolor Detection Set (Cat. No ). Just before use, add one colored substrate tablet to 10 ml of Detection Buffer to make Color Substrate Solution. AP inactivation buffer 50 mm EDTA Adjust ph to 8.0 (20 C). 208

5 5. Other Solutions and Buffers 5. Other Solutions and Buffers N-lauroylsarcosine stock solution Dissolve 10% (w/v) N-lauroylsarcosine in water. Filter through a membrane ( µm). High SDS hybridization buffer 7% SDS 50% formamide, deionized 2% Blocking Solution [1:5 dilution of stock (10%) Blocking Solution; see Section 4 above.] 50 mm sodium phosphate, ph 7.0 To prepare 500 ml of high SDS hybridization buffer, combine the following stock solutions in the order given below: 100% formamide, deionized 250 ml 30x SSC 83 ml 1 M sodium phosphate, ph ml 10% Blocking Solution 100 ml 10% N-lauroylsarcosine 5 ml Pour the resulting solution into an Erlenmeyer flask that contains 35 g SDS. (Caution: Do not inhale. Heat and stir the solution until the SDS dissolves. Adjust the total volume of the solution to 500 ml with sterile, double distilled water. Store the solution at room temperature, but heat to 65 C before use. Standard hybridization buffer 0.02% SDS 1% Blocking Solution [1:10 dilution of stock (10%) Blocking Solution] Standard hybridization buffer + 50% formamide 50% formamide, deionized 0.02% SDS 2% Blocking Solution [1:5 dilution of stock (10%) Blocking Solution] 209