Solutions

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Mol.biology // Next-generation sequencing

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Содержание


[править] Betaine, 5M

  • stabilizer of PCR reaction
  • code: {{:Solutions/Betaine, 5M}}

store

» solid Betaine at +4°C
» Betaine solution at -20°C

ρ = 1.073g/ml

monohydrate C5H11NO2xH2O; Mw=135.2g/M

conc.stock100ml200ml700ml
Betaine5M135.2g/M67.6g135.2g473.2g
H2O mQ39.74ml79.4ml277.9ml


anhydrous C5H11NO2; Mw=117.15g/M

conc.stock100ml200ml700ml
Betaine5M 117.15g/M58.58g117.15g410.03g
H2O mQ48.73ml97.49ml341.08ml


  • dissolve;
  • rotate at 37°C for 1h;
  • filter through 0.2µm filter (NB! wet filter with water before filtering 5M solution);
  • prepare 12ml aliquots in 50ml tubes;
  • store at -20°C (solution is a liquid at this temperature)



[править] EB, 10x

  • low-salt dilution buffer for NA
  • component of spin-column purification kits from Qiagen
  • code: {{:Solutions/EB, 10x}}

store at +4°C
pH8.5

conc.stock1ml5ml
Tris Cl, pH8.50.1M1M0.1ml0.5ml
H2O mQ900µl4.5ml

  • sterilize by autoclaving or filtration



[править] Ethanol, 70%

  • washing of NA pellet
  • code: {{:Solutions/Ethanol, 70}}

store at normal temperature in tightly closed bottle/tube
ρ = 0.853g/ml

conc.stock50ml250ml1L
Ethanol70% (v/v)100%, 0.79g/ml27.7g138g553g
H2O mQ15ml75ml300ml



[править] Instrument buffer, 1x

  • buffer for SOLiD sequencing machine
  • code: {{:Solutions/Instrument buffer, 1x}}

store at +4°C for 10 days
ρ = 1.054g/ml

ρ1L
H2O1g/ml700ml
10x Instrument buffer1.014g/ml100ml / 101.4g /
Glycerol1.262g/ml200ml / 252.4g /



[править] Library Storage buffer

  • buffer for storage of diluted NA solutions
  • code: {{:Solutions/Library Storage buffer}}

store at -20°C

conc.stock1ml5ml
Tween0.1%10%10µl50µl
EB  0.99ml4.95ml



[править] PEG 8000, 25%

  • molecular crowding agent for ligation
  • component of PEG/TAE elution buffer
  • code: {{:Solutions/PEG 8000, 25}}

store

» solid PEG at normal temperature
» solution at -20°C, snap freeze. NB! do not store solution at 0-4°C

ρ = 1.035g/ml

conc.stock400µl
PEG-800025% (w/v)solid100mg
H2O mQ314µl

  • prepare ~100mg aliquots and store them at NT
  • prepare immediately before use
  • for storage:
» make aliquots
» avoid multiple freeze/thawing



[править] SBB, 2x

  • buffer for binding of biotinilated molecules to streptavidin-coated beads
  • code: {{:Solutions/SBB, 2x}}

Streptavidin Binding Buffer, 2x
store at +4°C in ~1ml aliquots

conc.stock0.5ml1ml12ml
Tris-HCl, pH 7.510mM1M5µl10µl120µl
NaCl2M5M200µl400µl4.8ml
EDTA, pH 81mM0.5M1µl2µl24µl
H2O mQ294µl588µl7.06ml



[править] Sinefungin, 10mM

  • cofactor for EcoP15I restrictase. Without Sinefungin EcoP15I requires two sites with opposite orientation. It cuts at single site in presence of Sinefungin
  • code: {{:Solutions/Sinefungin, 10mM}}

store

» solid Sinefungin at +4°C
» Sinefungin solution at -20°C, snap freeze.

conc.stock  
Sinefungin10mM381.4g/M1mg
H2O mQ260µl

  • prepare ~1mg aliquots
  • prepare solution immediately before use



[править] SSC, 20x

  • blotting of NA
  • storage of FlowCells for Illumina sequencing machine
  • code: {{:Solutions/SSC, 20x}}

store at normal temperature
ρ = 1.103g/ml
pH should be ~7.0

conc.stock0.5L1.5L2L
NaCl3M58.44g/M87.66g263.0g350.6g
Na3C6H5O7x 5.5H2O0.3M357.1g/M53.57g160.7g214.3g
H2O mQ440.3ml1.32L1.76L



[править] STE, 10x

  • spectrophotometric analysis of NA
  • buffer for Phenol extraction of NA
  • code: {{:Solutions/STE, 10x}}

store at +4°C
pH7.5

conc.stock1ml5ml
NaCl1M5M0.2ml1.0ml
Tris Cl, pH7.50.1M1M0.1ml0.5ml
EDTA10mM0.5M20µl0.1ml
H2O mQ680µl3.4ml

  • sterilize by autoclaving or filtration




[править] TE, 10x

  • low-salt dilution buffer for NA
  • code: {{:Solutions/TE, 10x}}

store at +4°C
pH7.5

conc.stock1ml5ml
Tris Cl, pH7.50.1M1M0.1ml0.5ml
EDTA10mM0.5M20µl0.1ml
H2O mQ880µl4.4ml

  • sterilize by autoclaving or filtration



Источник — «http://www.molbiol.ru/wiki/Solutions»


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