DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols

Solutions

(last modified March 31, 2000)


Abbreviations


WFA01: 0.5 M (NH4)2SO4

  1. dissolve 6.61 g (NH4)2SO4 in 80 ml milliQ water
  2. add milliQ water to 100 ml
  3. autovclave
    NOTE: store up to 1 year at RT

WFA09: 10% APS

  1. take a vial with 25 g ammonium persulfate (APS) and dissolve it in 200 ml milliQ water
    NOTE: careful, APS is an oxidative substance
  2. add milliQ water to 250 ml
  3. divide into 10 ml portions in 10 ml blue-cap vials
    NOTE: store maximal 1 year at -20C or up to 1 month at 4C

WFA10: ALF loading mix

  1. dissolve 0.5 g blue dextran (D-5751, Sigma) in 100 ml de-ionized formamide (WFF01)
    NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
  2. warm to dissolve the blue dextran (D-5751, Sigma)
    NOTE: store up to 2 year at 4C

WFA11: 9% PAA, 80% UF, 0.5 x TAE

  1. dissolve 210 g urea in 6.3 ml 50 x TAE-buffer (WFT09), 140 ml 40% acrylamide/bis-acrylamide solution (37.5:1) and 200 ml de-ionized formamide (WFF01)
    NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
    NOTE: use new unopened 100 ml flasks formamide
  2. add milliQ water to 625 ml
    NOTE: store up to 3 months at 4C in a light-tight bottle

WFA12: 9% PAA, 0% UF, 0.5 x TAE

  1. add 225 ml 40% acrylamide/bis-acrylamide solution (37.5:1) to 10 ml 50 x TAE-buffer (WFT09)
  2. add milliQ water to 1000 ml
    NOTE: store up to 1 year at 4C in a light-tight bottle

WFB01: 20 x bloodlysis buffer (3.1 M NH4Cl, 0.2 M KHCO3, 20 mM EDTA, pH7.4)

  1. dissolve 331 g NH4Cl and 40 g KHCO3 in 1.6 l milliQ water
  2. add 14.8 g EDTA disodium salt and dissolve by heating
  3. adjust pH to 7.4 with 5.0 M NaOH (WFN04)
  4. add milliQ water to 2 l
  5. autoclave
    NOTE: store up to 1 year at RT

NOTE: test the buffer on a spare blood sample (according to ALG001); when a proper leucocyte pellet is obtained the buffer may be used for regular DNA-isolations


WFB04: 10% blocking buffer

  1. dissolve 50 g Blocking reagens (#1096176, Boehringer) in 300 ml milliQ water and 50 ml 10 x maleic acid (WFM02)
  2. add milliQ water to 500 ml
  3. autoclave
  4. distribute 8 ml aliquots in 14 ml white-cap tubes.
    NOTE: store max 2 years at -20C

WFE01: 10 mM EDTA

  1. dilute 0.5 M EDTA pH8.0 (15575-012, Life Technologies) 50 times; take 98 ml milliQ water and add 2 ml 0.5 M EDTA pH8.0
  2. autoclave
    NOTE: store up to 1 year at RT

WFF01: de-ionized formamide (ABI-310 fragmentanalysis)

  1. take a cilinder and add 25 ml (de-ionized) formamide and 1 g mixed bed resin (AG 501X8)
    NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
  2. close the cilinder with parafilm and mix 10 min
  3. measure the pH using a pH paper
    NOTE: pH must be at least 7.0, but a pH of about 9.0 is optimal. If necessary, repeat the de-ionization step until the desired pH is reached
  4. let the resin settle down
  5. distribute 500 ul aliquots de-ionized formamide into 1.5 ml Eppendorf tubes and store -20C
    NOTE: store up to 1 year at -20C

WFH01: hyb-mix (NaPi/SDS/PEG)

  1. freshly prepare 1 l NaPi-buffer;
    1. dissolve 88.9 g Na2HPO4.2H2O in 900 ml milliQ water
    2. adjust the pH to 7.2 using 85% H3PO4
    3. add milliQ water to 1 l
      NOTE: prepare fresh, do not store
  2. take 1 l NaPi-buffer, add 200 ml 5.0 M NaCl (WFN01) and 8 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies)
  3. add 280 g SDS (Sigma) and dissolve
    NOTE: to weigh SDS, work in a fume hood, wear gloves, a lab coat and a mouth protection
  4. add 400 g PEG-6000 and dissolve
    NOTE: if necessary, warm slightly to dissolve
  5. add milliQ water to 4 l
  6. distribute over 2 flasks of 2 l
    NOTE: store up to 6 months

NOTE: the quality of the hyb-mix should be tested following protocol ALG005 to label a good probe and hybrizing it to an older, working Southern blot. When the signal obtained is OK, the hyb-mix may be used for regular experiments 0.125 M Na2HPO4 (pH7.2 with H3PO4), 0.25 M NaCl, 1.0 mM Na2EDTA, 7% SDS (Sigma, 44244), 10% PEG-6,000 (BDH Chemicals Ltd., 44271)


WFK01: nucleus lysis buffer (10 mM M Tris-HCl, 0.4 M NaCl, 2 mM EDTA, pH8.2)

  1. dissolve 2.42 g Tris in 1.6 l milliQ water
  2. add 8 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies) and 160 ml 5.0 M NaCl (WFN01)
  3. adjust pH to 8.2 with HCl (use glass pipet)
  4. add milliQ water to 2 l
    NOTE: store up to 3 months at RT

WFK02: 1.0 M KCl

  1. dissolve 7.45 g KCl in 80 ml milliQ water
  2. add milliQ water to 100
  3. autoclave
    NOTE: store up to 1 year at RT

WFK03: 3.0 M KCl

  1. dissolve 18.6 g KCL in 200 ml milliQ water
  2. add milliQ water to 250 ml
  3. autoclave
    NOTE: store up to 1 year at RT

WFL01: lye-salt solution

  1. dissolve 2.4 g NaOH in 150 ml milliQ water
  2. add 4 ml 5.0 M NaCl (WFN01)
  3. add milliQ water to 200 ml met milliQ water
    NOTE: store up to 1 month at RT

WFL03: 2 x SSCP/STR loading mix

  1. take 95 ml (de-ionized) formamide (WFF01)
    NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
  2. add 50 mg bromophenolblue (B7021, Sigma) and 50 mg xylene cyanol (XC)
  3. add 4 ml 0.50 M EDTA pH8.0 (15575-012, Life Technologies) and mix until both colour stains have dissolved
    NOTE: store up to 2 year at 4C

WFL04: bacteriophage lambda DNA dilution range

  1. prepare a 50 ng/l bacteriophage lambda DNA dilution by adding 40 l lambda DNA (stock is 500 ng/l) to 360 l autoclaved milliQ water
  2. make a dilution range according to the schema below (start with pipetting the SLM-buffer [WFS03] and milliQ water, add lambda DNA last);
    Concentration l lambda DNA dilution (50 ng/l) l autoclaved
    milliQ water
    l 2 x SLM-buffer
    1.0 ng/l 10 240 250
    2.5 ng/l 25 225 250
    5.0 ng/l 50 200 250
    10 ng/l 100 150 250
    20 ng/l 200 50 250
  3. mark the concentration and preparation date on a sticker on the Eppendorf tubes
    NOTE: store up to 1 year at 4C

WFM01: 1.0 M MgCl2

  1. dissolve 20.33 g MgCl2.6H2O in 80 ml milliQ water
  2. add milliQ water to 100 ml
  3. autoclave
    NOTE: store up to 1 year at RT

WFM02: 10 x maleic acid

  1. dissolve 116 g maleic acid (1.0 M) and 87.7 g NaCl (1.5 M) in 900 ml milliQ water
  2. adjust the pH to 7.5 with solid NaOH (80-100 g)
    NOTE: the initial white precipitate dissolves at pH7.5
    NOTE: store up to 1 year at 4C

WFM03: GeneRuler DNA ladder mix (50 ng/l length marker; 100 bp ladder)

  1. add 125 l DNA ladder mix (0.40 g/l) to 775 l milliQ water
  2. take an Eppendorf tube with 10 x SLM-buffer (WFS03), warm until the SDS dissolves and add 100 l to the diluted DNA ladder mix
  3. distribute over two Safe-Lock tubes
    NOTE: store up to 1 year at 4C

WFM04: bacteriophage lambda HindIII+PstI marker

  1. perform two bacteriophage lambda restrition enzyme digestions;
    1. take 500 g lambda DNA (1.0 ml of a 500 g/ml stock)
    2. 120 l 10 x R+ buffer (MBI)
    3. tube 1,  500 U HindIII
      tube 2,  500 U PstI
    4. add milliQ water to 1.2 ml endvolume
  2. incubate 4 h at 37C
  3. take 1 l digested DNA and check on a 0.8 % agarose gel (minimal migration ~10 cm)
    NOTE: if the difestion is incomplete, add 100 U enzym and incubate again 4 h at 37C
  4. if digestion is complete, mix the samples and dilute 10 times in 1 x SLM-buffer, i.e.;
    1. warm a tube with 10 x SLM-buffer (WFS03) to dissolve precipitated SDS
    2. add 1.0 ml 10 x SLM-buffer to the DNA-mixture
    3. add milliQ water to 10.0 ml (i.e. add about 6.6 ml)
  5. check the new sample by loading 10 l on a 0.8 % agarose gel, next to 10 l of the previously prepared sample
    NOTE: if necessary, modify the dilution
  6. distribute the digested samples in 1.0 ml aliquots in marked Safe-Lock tubes
    NOTE: store up to 5 year at -20C (post-PCR lab)

WFM06: invisible bacteriophage lambda HindIII and PstI marker

  1. dilute a bacteriophage lambda DNA HindIII+PstI marker (prepared according to WFM04) to 2 ng/l;
    1. take 100 l lambda DNA HindIII+PstI marker
    2. add 0.5 ml 10 x SLM-buffer (WFS03)
    3. add milliQ water to 5.0 ml (i.e. add about 4.4 ml)
  2. use 10 l as marker on a agarose gel for Southern blotting (ALG005)
  3. to detect the DNA marker, hybridize with labeled lambda DNA as described (ALG005)
  4. distribute the digested samples in 1.0 ml aliquots in marked Safe-Lock tubes
    NOTE: store up to 5 year at -20C (post-PCR lab)

WFN01: 5.0 M NaCl

  1. dissolve 292.2 g NaCl in 800 ml milliQ water
  2. add milliQ water to 1 l
  3. autoclave
    NOTE: store up to 1 year at RT

WFN02: 6.0 M NaCl

  1. dissolve 350.64 g  NaCl in 800 milliQ water (is saturated)
  2. add milliQ water to 1 l
  3. autoclave
    NOTE: store up to 1 year at RT

WFN03: 3.0 M NaAc (pH 5.6)

  1. dissolve 408.1 g NaAc.3H2O (sodium acetate) in 800 ml milliQ water
  2. adjust pH to 5.6 with acetic acid (glacial HAc, about 40 ml, use glass pipet)
  3. add milliQ water to 1.0 l
  4. re-adjust pH to 5.6 with acetic acid
  5. autoclave
    NOTE: store up to 1 year at RT

WFN04: 5 N NaOH

  1. dissolve 10 g NaOH in 40 ml milliQ water in a blue-cap tube
  2. add milliQ water to 50 ml
    NOTE: store up to 1 month at RT

WFN06: 2 mM dNTP mix (2 mM dATP, dCTP, dGTP and dTTP)

  1. pipet 11.5 ml autoclaved milliQ water in a 15 ml tube
    NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
  2. add 150 l of each dNTP
  3. mix carefully and distribute 1 ml aliquots to marked 1.5 ml Safe-Lock tubes
    NOTE: store up to 1 year at -20C

WFN07: 25 mM dNTP mix (25 mM dATP, dCTP, dGTP and dTTP)

  1. pipet 250 l of each dNTP into a 1.5 ml Safe-Lock tube
    NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
  2. mix carefully and distribute 100 l aliquots to marked 1.5 ml Safe-Lock tubes
    NOTE: store up to 1 year at -20C

WFN08: dNTP mix for radio-active PCR’s (2 mM dATP, dGTP and dTTP, 25 M dCTP)

  1. pipet 11.7 ml autoclaved milliQ water in a 15 ml tube
    NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
  2. add 250 l dATP, dGTP and dTTP solution and 3.1 l dCTPsolution
  3. mix carefully and distribute 1.0 ml aliquots to marked 1.5 ml Safe-Lock tubes
    NOTE: store up to 1 year at -20C

WFP01: pronase (20 mg/ml)

  1. dissolve 1 g pronase (165921, Boehringer Mannheim GmbH) in 50 ml milliQ water (blue-cap Greiner tube)
  2. incubate (self digest) 1 h by 37oC
  3. distribute 1.0 ml aliquots to 1.5 ml Safe-Lock tubes
    NOTE: store up to 1 year at -20C
    NOTE: never refreeze after thawing

WFP02: 5 x multiplex or STR PCR-buffer

  1. the 5 x PCR-buffer has final concentrations of 83 mM (NH4)2SO4, 335 mM Tris-HCl pH8.8, 33.5 or 7.5 mM MgCl2, 34 M EDTA and 50 mM beta-mercapto ethanol. To prepare the buffers, add to a blue-cap tube;
      multiplex-
    buffer
    STR-
    buffer
    0.5 M (NH4)2SO4 8.3 ml 8.3 ml
    0.5 M Tris-HCl pH8.8 33.5 ml 33.5 ml
    1.0 M MgCl2 1.675 ml 0.375 ml
    10 mM EDTA 170 l 170 l
    14 M beta-mercapto-ethanol 179 l 179 l
    autoclaved milliQ water 6.2 ml 7.5 ml
  2. distribute 1 ml aliquots to marked 1.5 ml Safe-Lock tubes
    NOTE: store up to 2 year at -20C

WFP06: 10 x PBS (0.137 mM NaCl / 2.7 mM KCl /8.0 mM Na2HPO4 / .. mM KH2PO4, non radio-active PTT)

  1. dissolve in 1.9 l milliQ water
  2. adjust the pH to 7.5 with 5 N NaOH (i.e. about 10 - 15 ml)
  3. add milliQ water to 2 l
  4. autoclave
    NOTE: store up to 1 year at RT

WFP07: 6 x PAAGE loading buffer

  1. dissolve in 75 ml milliQ water;
  2. add milliQ water to 100 ml
    NOTE: store up to 2 year at RT

WFS02: 10 x SE-buffer (0.75 M NaCl, 0.24 M EDTA, pH8.0)

  1. dissolve 43.8 g NaCl and 89.3 g EDTA disodium salt in 800 ml milliQ water
  2. add 11 g NaOH and dissolve
  3. adjust pH to 8.0 with 5 N NaOH
  4. add milliQ water to 1 l
  5. autoclave
    NOTE: store up to 1 year at RT

WFS03: 10 x SLM-buffer

  1. dissolve 40 g sucrose (10274 5C, BDH) in 20 ml milliQ water
  2. add;
  3. add milliQ water to 100 ml
  4. add 250 mg bromophenolblue (B7021, Sigma) and dissolve
    NOTE: store up to 2 year at RT

WFS04: 1.0 M spermidine stock

  1. dissolve in a white-cap tube 1 g spermidine (S 0266, Sigma) in 3.0 ml autoclaved milliQ water
  2. add milliQ water to 6.9 ml (= 1.0 M stock solution)
  3. distribute 1.0 ml aliquots to marked 1.5 ml Safe-Lock tubes
    NOTE: store up to 5 year at -20C
    NOTE: for 100 mM spermidine take 1 ml 1.0 M spermidine stock, add milliQ water to 10 ml and distribute 200 l aliquots to marked 1.5 ml Safe-Lock tubes

NOTE: spermidine is agressive and causes burns. Wear protective clothing (lab coat), gloves and protect your eyes. When your eyes come into contact with spermidine, immediately wash with plenty of water and ask for medical advise. Immediately remove any contaminated clothing (see also MSDS)


WFS06: 2 x stopmix

  1. weigh 200 mg blue dextran (D-5751, Sigma) and 20 mg phenol red (phenolsulfonphtalein) (P-4758, Sigma)
  2. dissolve in 95 ml milliQ water
  3. add 5 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies)
    NOTE: store up to 4 year at RT

WFS08: 10 x Syl PCR-buffer

  1. mix in a sterile blue cap tube;
  2. distribute 1 ml aliquots in 1.5 ml Safe-Lock tubes
    NOTE: store up to 2 year at -20C

WFT02: TE-4 (10 mM M Tris-HCl, 0.1 mM EDTA, pH7.5)

  1. add 5 ml 1.0 M Tris.HCl pH7.5 (WFT04) and 100 l 0.5 M EDTA pH 8.0 (15575-012, Life Technologies) to 400 ml milliQ water
  2. add milliQ water to 500 ml
  3. distribute over 5 bottles with 100 ml each
  4. autoclave
    NOTE: store up to 1 year at RT

WFT04: Tris-HCl (0.5 / 1.0 / 1.5 M; pH adjusted)

  1. dissolve the required amount of Tris in 80 ml milliQ water;
  2. adjust the pH to required figure using HCl (36-38 %), use a glass pipet
  3. add milliQ water to 100 ml
  4. autoclave
    NOTE: store up to 1 year at 4C

WFT08: 10 x transferbuffer (non radio-active PTT)

  1. dissolve in 1.5 l milliQ water
  2. add milliQ water to 2 l
    NOTE: store up to 1 year at 4C

WFT09: 50 x TAE-buffer

  1. dissolve in 1.0 l milliQ water
  2. adjust the pH to 8.0 with acetic acid (about 150 ml)
  3. add milliQ water to 2 l
  4. autoclave
    NOTE: store up to 1 year at RT

WFW01: autoclaved milliQ water

  1. fill special 200 ml black capped flasks with milliQ water
  2. autoclave
  3. mark the flasks with date, content, initials and max. storage date
    NOTE: store up to 1 year at RT
    NOTE: each flask may be opened once during up to 1 year to fill 1.5 ml Safe-Lock tubes


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