Yu:Northern solns: Difference between revisions

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=Northern Solutions=
=Northern Solutions=


===1.3% Agarose Formaldehyde gel(85 mL)===
#Dissolve 1.16 g of agarose in 62mL of milliQ H<sub>2</sub>O
#Cool agarose to 55-60 °C in a pre-warmed water bath
#Add 8.5 mL of [[Yu:Northern_solns#10x_MOPs|10x MOPs]]
#Add 14.45 mL of 37% formaldehyde in the hood
#Pour gel immediately and allow to cool of at least 30 min in the hood
===20x SSC===
{| border="1" cellpadding="10" cellspacing="0"
{| border="1" cellpadding="10" cellspacing="0"
|!'''20x <span id="SSC">SSC</span>'''
|width="300px" valign="top"|'''Final Concentration'''
|width="300px" valign="top"|'''Final Concentration'''
|width="100px" valign="top"|'''For 1 Liter'''
|width="100px" valign="top"|'''For 1 Liter'''
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|}
|}


 
===10x MOPs===
{| border="1" cellpadding="10" cellspacing="0"
{| border="1" cellpadding="10" cellspacing="0"
|!'''<span id="10x MOPS">10x MOPS</span>'''
|width="300px" valign="top"|'''Final Concentration'''
|width="300px" valign="top"|'''Final Concentration'''
|width="100px" valign="top"|'''For 500 mL'''
|width="100px" valign="top"|'''For 500 mL'''
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|colspan="3"|Store in the Dark at 4 °C
|colspan="3"|Store in the Dark at 4 °C
|}
|}
===100x Denhardt's===
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="100px" valign="top"|'''For 100 mL'''
|width="100px" valign="top"|'''For 500 mL'''
|-
|Ficoll 400
|2 g
|10 g
|-
|Polyvinylpyrrolidone MW 360000
|2 g
|10 g
|-
|BSA fraction V
|2 g
|10 g
|-
|colspan="3"|milliQ H<sub>2</sub>O
|-
|colspan="3"|Store at -20 °C
|}
===Hybridization Buffers===
====Oligo (Pre)Hybridization Buffer====
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="200px" valign="top"|'''For 100 mL'''
|width="200px" valign="top"|'''For 200 mL'''
|-
|3x SSC
|15 mL of [[Yu:Northern_solns#20x_SSC|20x SSC]]
|30 mL of [[Yu:Northern_solns#20x_SSC|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 H<sub>2</sub>
|to 100 mL
|to 200 mL
|-
|}
====Hyb Buffer====
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="200px" valign="top"|'''For 50 mL'''
|width="200px" valign="top"|'''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 NaPO<sub>4</sub> Buffer pH 7.0
|5 mL of 1M NaPO<sub>4</sub> 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 H<sub>2</sub>
|to 50 mL
|to 100 mL
|-
|}
===Oligo Wash Buffer===
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="200px" valign="top"|'''For 500 mL'''
|width="200px" valign="top"|'''For 1 L'''
|-
|2x SSC
|50 mL of [[Yu:Northern_solns#20x_SSC|20x SSC]]
|100 mL of [[Yu:Northern_solns#20x_SSC|20x SSC]]
|-
|0.1% SDS
|5 mL of 10% SDS
|10 mL of 10% SDS
|-
|milliQ H<sub>2</sub>
|to 500 mL
|to 1 L
|-
|}
===Loading Buffers===
====MOPs Loading Buffer====
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="200px" valign="top"|'''For 1 mL'''
|width="200px" valign="top"|'''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 H<sub>2</sub>
|215 μL
|2.15 mL
|-
|colspan="3"|pinch of Bromophenol Blue
|-
|colspan="3"|Store in the dark at -20 °C
|}
====Formamide Loading Buffer====
{| border="1" cellpadding="10" cellspacing="0"
|width="300px" valign="top"|'''Final Concentration'''
|width="200px" valign="top"|'''For 1 mL'''
|width="200px" valign="top"|'''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
|-
|colspan="3"|Pinch of Xylene Cyanol and Bromophenol Blue
|-
|colspan="3"|'''Store in the dark at -20 °C'''
|}
[[Category:Yu Solutions]]

Latest revision as of 13:57, 22 September 2009

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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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