Yu:Northern solns

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Contents

Northern Solutions

1.3% Agarose Formaldehyde gel(85 mL)

  1. Dissolve 1.16 g of agarose in 62mL of milliQ H2O
  2. Cool agarose to 55-60 °C in a pre-warmed water bath
  3. Add 8.5 mL of 10x MOPs
  4. Add 14.45 mL of 37% formaldehyde in the hood
  5. Pour gel immediately and allow to cool of at least 30 min in the hood

20x SSC

Final Concentration For 1 Liter For 4 Liters
3M NaCl 175.3 g 701.2 g
0.3M NaCitrate 88.2 g 352.8 g
milliQ H2O to 1 L to 4 L

10x MOPs

Final Concentration For 500 mL For 1 Liter
0.4 M Morpholinopropanesulfonic (MOPS) acid (free acid) 41.9 g MOPS 83.8 g MOPS
0.1 M Na-acetate-3 x H2O 6.8 g 13.6 g
10 mM EDTA 10 mL 0.5 M EDTA 20 mL 0.5 M EDTA
Adjust pH to 7.2 with NaOH
Store in the Dark at 4 °C

100x Denhardt's

Final Concentration For 100 mL For 500 mL
Ficoll 400 2 g 10 g
Polyvinylpyrrolidone MW 360000 2 g 10 g
BSA fraction V 2 g 10 g
milliQ H2O
Store at -20 °C

Hybridization Buffers

Oligo (Pre)Hybridization Buffer

Final Concentration For 100 mL For 200 mL
3x SSC 15 mL of 20x SSC 30 mL of 20x SSC
0.5% SDS 5 mL of 10% SDS 10 mL of 10% SDS
1x Denhardt's 2 mL of 50x Denhardt's 4 mL of 50x Denhardt's
100 μg/mL ssDNA 1 mL 10 mg/mL ssDNA 2 mL 10 mg/mL ssDNA
milliQ H2 to 100 mL to 200 mL

Hyb Buffer

Final Concentration For 50 mL For 100 mL
PIPES 2.5 mL of 1M PIPES pH 6.5 5 mL of 1M PIPES pH 6.5
Sodium Phosphate Buffer 2.5 mL of 1M NaPO4 Buffer pH 7.0 5 mL of 1M NaPO4 Buffer pH 7.0
Sodium Chloride 1 mL of 5M NaCl 2 mL of 1 mL of 5M NaCl
SDS 12.5 mL of 20% SDS 25 mL of 20% SDS
EDTA 250 μL of 0.5M EDTA pH 8.0 500 μL of 0.5M EDTA pH 8.0
ssDNA 300 μL 10 mg/mL ssDNA 600 μL 10 mg/mL ssDNA
milliQ H2 to 50 mL to 100 mL

Oligo Wash Buffer

Final Concentration For 500 mL For 1 L
2x SSC 50 mL of 20x SSC 100 mL of 20x SSC
0.1% SDS 5 mL of 10% SDS 10 mL of 10% SDS
milliQ H2 to 500 mL to 1 L

Loading Buffers

MOPs Loading Buffer

Final Concentration For 1 mL For 10 mL
1x MOPs 100 μL 1 mL
18.5% Formaldehyde 185 μL 1.85 mL
50% Formamide 500 μL 5 mL
4% Ficoll 400 40 mg 400 mg
milliQ H2 215 μL 2.15 mL
pinch of Bromophenol Blue
Store in the dark at -20 °C

Formamide Loading Buffer

Final Concentration For 1 mL For 10 mL
>99% Formamide 0.96 mL of deionized Formamide 9.6 mL of deionized Formamide
20 mM EDTA 4 μL of 0.5M EDTA 400 μL of 0.5M EDTA
Pinch of Xylene Cyanol and Bromophenol Blue
Store in the dark at -20 °C
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