常用试剂配制

互联网2013-09-05

4660

TE 1x, 10x
STE 1x, 10x
SSC 20x
Electrophoresis buffers.

TAE 50x
TBE 10x
FGRB 50x
GTGB 20x
TGB 5x

Loading buffer 10x
PEG-NaCl
Bacteriophage lambda buffers

SM
STM
TM

Denaturation buffer
Neutraliszation buffer
Developer Rentgen-2
Fixer BKF-2
Scintillation solution
Plasmid solution I
Plasmid solution II
Oligonucleotide gel staining mix
2M Mg²⁺ solution
Hybridization buffer HSB (f⁺ )
Restriction buffers

А., B., L., M., H.

Denhardt solution 50х
PBS 10x
SSPE 20x
RNase (DNase free)

Proteinase K solution
EDTA/glycogen solution 20x
DNase RQ1 buffer

Supplement.

<center><font>About the recalculation of recipes for the arbitrary volume:<br /> <br /> </font> </center>

1x; pH7.4; 7.6; 8.0; (store at 4^o C).

	Conc.	Stock	1ml	5ml	10ml	50ml	400ml
Tris Cl	10mM	1M	10µl	50µl	100µl	500µl	4ml
EDTA	1mM	0.5M	2µl	10µl	20µl	100µl	800µl
H₂ O		mQ	988µl	4.94ml	9.88ml	49.4ml	395ml

10x:

	Conc.	Stock	1ml	5ml
Tris Cl	0.1M	1M	0.1ml	0.5ml
EDTA	10mM	0.5M	20µl	0.1ml
H₂ O	mQ		880µl	4.4ml

sterilize by autoclaving

STE

1x; (store at 4^o C).

	Conc.	Stock	1ml	5ml	10ml	50ml
NaCl	0.1M	5M	20µl	100µl	200µl	1.0ml
Tris Cl, pH8.0	10mM	1M	10µl	50µl	100µl	0.5ml
EDTA, pH8.0	1mM	0.5M	2µl	10µl	20µl	100µl
H₂ O		mQ	0.968ml	4.84ml	9.68ml	48.4ml

10x:

	Conc.	Stock	1ml	5ml
NaCl	1.0M	5M	0.2ml	1.0ml
Tris Cl	0.1M	1M	0.1ml	0.5ml
EDTA	10mM	0.5mM	20µl	0.1ml
H₂ O	mQ		680µl	3.4ml

sterilize by autoclaving

SSC

20x; pH 7.0; (store at NT).

	Conc.	Stock	0.5L	1.5L	2L
NaCl	3M	58.44g/M	87.66g	263.0g	350.6g
Na₃ C₆ H₅ O₇ x 5.5H₂ O	0.3M	357.1g/M	53.57g	160.7g	214.3g
H₂ O		mQ	440.3ml	1.32L	1.76L

p = 1.103

TAE

1x pH = 7.6:

Tris-acetate 40mM
EDTA 1mM

store the main stock at 4^o C, working quantity (~100ml) at NT

	Conc.	Stock	100ml	150ml	200ml	250ml
Tris-base	2M	121.1g/M	24.22g	36.33g	48.44g	60.55g
EDTA	0.05M	372.3g/M	1.862g	2.792g	3.723g	4.654g
Acetic acid	~1.56M	17.4M	~8.96ml ~9.40g	~13.44ml ~14.10g	~17.92ml ~18.80g	~22.40ml ~23.50g	~ ~
H₂ O		mQ	~73.30g	~109.95g	~146.60g	~183.25g	~

p(50x)=1.0878

it is convenient to solubilize Tris and EDTA first then adjust pH by acetic acid.

TBE

10x, pH ~8.3;(store at NT in clean bottle)

	1x	10x	Stock	1L
Tris base	89mM	0.89M	121.14g/M	107.8g
Boric acid	89mM	0.89M	61.83g/M	55.03g
EDTA, pH 8.0	2mM	20mM	500mM	40ml 43.84g
H₂ O			mQ	863.3ml

10x stock solution is stable at NT after 0.4µm filtration;
for PAAG use as 1x, for agarose gel - as 0.5x;
TBE is used in agarose gels for separation of small (< 200bp) fragments. Purification of fragments from TBE-based gels is more complicated than from TAE-based gels.

FGRB

50x (formaldehyde gel-running buffer), p=1.104g/ml (store at +4^o C):

	Conc.	Stock	0.5L	1.0L	2.0L
MOPS (pH7.0)	1.0M	209.27g/M	104.64g	209.27g	418.54g
AcONax3H₂ O	0.5M	136.08g/M	34.02g	68.04g	136.08g
EDTAx2H₂ O	50mM	372.3g/M	9.31g	18.62g	37.23g
H₂ O		mQ	404.0ml	808ml	1616ml

	Conc.	Stock	0.5L	1.0L	2.0L
MOPS (pH7.0)	1.0M	209.27g/M	104.64g	209.27g	418.54g
AcONa anhydrous	0.5M	82.04g/M	20.51g	41.02g	82.04g
EDTA x 2H₂ O	50mM	372.3g/M	9.31g	18.62g	37.23g
H₂ O		mQ	417.5ml	835ml	1670ml

adjust pH7.0 10N NaOH (per 500ml of buffer ~22ml), filter-sterilize through 0.22µm filter;
solution becomes yellow during the storage on the light, but it has no effect on the solution quality.

GTGB

20x /Glycerol tolerant gel buffer/ (store at +4^o C):

	Conc.	Stock	1L	1.5L	2.0L	2.5L
Tris base	1.78M	121.14g/M	216g	324g	432g	540g
Taurine	0.56M	125.15g/M	72g	108g	144g	180g
Na₂ EDTAx2H₂ O	10.7mM	372.3 g/M	4g	6g	8g	10g
H₂ O		mQ	802ml	1203ml	1604ml	2005ml

p=1.094

Taurine: C₂ H₇ NO₃ S; Mw=125.15g/M.

TGB

Tris-Glycine buffer 5x, pH8.3, (store the main stock at +4^o C, working quantity at NT).

	1x	Conc.	Stock	1L	1.5L
Tris-base	25mM	125mM	121.1g/M	15.1g	22.71g
Glycine	250mM	1.25M	75.05g/M	94g	141g
SDS	0.1%	0.5%	10%	50ml	75ml
H₂ O			mQ

Loading buffer

10x, (store at NT).

	Conc.	Stock	1ml	5ml	10ml	25ml
Xilene cyanol	0.1%	solid	1.0mg	5.0mg	10mg	25.0mg
Bromph. blue	0.1%	solid	1.0mg	5.0mg	10mg	25.0mg
SDS	0.5%	10%	50µl	250µl	500µl	1.25ml
EDTA, pH8.0	0.1M	0.5M	0.2ml	1.0ml	2.0ml	5.0ml
Glycerol	50%	100%	0.5ml 0.625g	2.5ml 3.125g	5.0ml 6.25g	12.5ml 15.63g
H₂ O		mQ	0.25ml	1.25ml	2.5ml	6.25ml

PEG-NaCl

(store at 4^o C).

	Conc.	Stock	50ml	100ml	150ml	200ml
NaCl	1.6M	58.44g/M	4.675g	9.35g	14.03g	18.70g
PEG-6000	13%	solid	6.50g	13.00g	19.50g	26.00g

or:

	Conc.	Stock	50ml	100ml	150ml	200ml
NaCl	1.6M	5M	16ml 18.97g	32ml 37.95g	48ml 56.92g	64ml 75.90g
PEG-6000	13%	40%	16.25ml	32.50ml	48.75ml	65.00ml

(store at 4^o C).

	Conc.	Stock	10ml	50ml	100ml	150ml
NaCl	0.1M	5M	200µl	1.0ml	2.0ml	3.0ml
MgSO₄~undefined	10mM	1M	100µl	0.5ml	1.0ml	1.5ml
Tris Cl, pH7.5	50mM	1M	0.5ml	2.5ml	5.0ml	7.5ml
	0.01%	2%	50µl	0.25ml	0.5ml	0.75ml
H₂ O		mQ	9.15ml	45.75ml	91.5ml	137.25ml

sterilize by autoclaving

STM

(store at 4^o C):

	Conc.	Stock	50ml
NaCl	10mM	5M	100µl
Tris Cl pH8.0	50mM	1M	2.5ml
MgCl₂	10mM	1M	500µl
H₂ O		mQ	46.9ml

(store at 4^o C):

	Conc.	Stock	50ml
Tris Cl pH8.0	50mM	1M	2.5ml
MgCl₂	10mM	1M	500µl
H₂ O		mQ	47.0ml

Denaturation buffer

(store at NT).

	Conc.	Stock	100ml	150ml	300ml	500ml	750ml
NaCl	1.5M	58.44g/M	8.766g	13.15g	26.30g	43.83g	65.75g
NaOH	0.5M	10M	5ml 6.65g	7.5ml 9.97g	15ml 19.94g	25ml 33.23g	37.5ml 49.84g
		40g/M	2g	3g	6g	10g	15g

Neutralization buffer

(store at NT).

	Conc.	Stock	150ml	300ml	500ml	750ml
Tris Cl, pH7.4	1M	121.1g/M	18.15g	36.3g	60.5g	90.75g
NaCl	1.5M	58.44g/M	13.15g	26.30g	43.83g	65.75g
HCl	~0.81M	11.6M/L	~10.5ml	~21ml	~35ml	~52.5ml
EDTA (optional)	1mM	0.5M	0.3ml	0.6ml	1ml	1.5ml

Developer Rentgen-2

(store at NT in the dark).

	500ml	800ml	1l
	1.1g	1.76g	2.2g
Na₂ SO₃ anhidr.	36.0g	57.6g	72.0g
Hydroquinone	4.4g	7.04g	8.8g
Na₂ CO₃	24.0g	38.4g	48.0g
KBr	2.0g	3.2g	4.0g
H₂ O	490ml	784ml	980ml

solubilize in the presented order , p=1.115g/ml.

Fixer BKF-2

(store at NT).

	500ml	800ml	1l
Na₂ S₂ O₃ x 5H₂ O	130g	208g	260g
NH₄ Cl	25g	40g	50g
Sodium pyrosulfite	8.5g	13.6g	17g
H₂ O	398ml	636.8ml	796ml

solubilize in the presented order, p=1.123g/ml.

Scintillation solution

(store at NT in the dark):

	0.5L	1L
PPO	2.50g	5.00g
POPOP	0.05g	0.10g
Toluene

Plasmid solution I

(store at +4^o C)

	Conc.	Stock	50ml	100ml	150ml	200ml
Glucose	50mM	2M	1.25ml 1.41g	2.5ml 2.825g	3.75ml 4.24g	5.0ml 5.56g
Tris Cl, pH 8.0	25mM	1.0 M	1.25ml	2.5ml	3.75ml	5.0ml
EDTA	10mM	0.5 M	1.0ml	2.0ml	3.0ml	4.0ml
H₂ O		mQ	46.5ml	93ml	139.5ml	186ml

Plasmid solution II

	Conc.	Stock	0.5ml	1ml	2.5ml	5.0ml	6.5ml	8.5ml
NaOH	0.2N	1N	0.1ml	0.2ml	0.5ml	1.0ml	1.3ml	1.7ml
SDS	1%	10%	0.05ml	0.1ml	0.25ml	0.5ml	0.65ml	0.85ml
H₂ O		mQ	0.350ml	0.7ml	1.75ml	3.5ml	4.55ml	5.95ml

Oligonucleotide gel staining mix

(store at NT).

	Conc.	Stock	100ml	200ml	300ml	500ml
AcONa(5.2)	0.5M	3M	17ml	33ml	50.0ml	83ml
Met. blue	0.04%	0.2%	20ml	40ml	60ml	100ml
H₂ O		mQ	73ml	127ml	190ml	317ml

Mg²⁺ 2M

(store at NT).

	Conc.	Stock	200ml
MgCl₂ x6H₂ O	1M	203.3g/M	40.66g
MgSO₄ x7H₂ O	1M	246.48g/M	49.30g
H₂ O		mQ	144.6g

p=1.173g/ml.

Hybridization buffer HSB (f⁺ )

high SDS hybridization buffer (store the main stock at 4^o C, working - NT)

	Conc.	Stock	500ml
SDS	7%	solid	35g
Formamid	50%	100%	250ml 283.3g
SSC	5x	20x	125ml 145.3g
Bloc. Reagent	2%	solid	10g
NaP (pH 7.0)	50mM	1M	25ml 27.6g
N-Lauroylsarcosine Na	0.1%	10%	5ml 5.1g
Salm.Sperm DNA	50µg/ml	10µg/µl	2.5ml
H₂ O		mQ	49ml

p=1.111

Blocking Reagent - is the powder from "Roche Diagnostics GmBH; former Boehringer Mannheim" #1096176. Instead of 2% Blocking Reagent one may use 10x Denhardt or 2% non-fat dry milk.
it is possible to dissolve the Blocking Reagent only in high strength buffer (in this particular case in H₂ O+SSC+NaP).
Salmon sperm DNA should be shredded and denaturated.

Restriction buffers 10x

А.

	Conc.	Stock	100µl	1ml
Tris Ac (pH7.9 37^o C)	33mM	1M	33µl	330µl
MgAc	10mM	1M	10µl	100µl
KAc	66mM	5M	13.2µl	132µl
DTT	0.5mM	1M	0.5µl	5µl
H₂ O		mQ	43.3µl	433µl

	Conc.	Stock	100µl	1ml
Tris Cl (pH 8.0 37^o C))	10mM	1M	10µl	100µl
MgCl₂	5mM	1M	5µl	50µl
NaCl	100mM	5M	20µl	200µl
b-Me	1mM	1M	1µl	10µl
H₂ O		mQ	64µl	640µl

	Conc.	Stock	100µl	1ml
Tris Cl (pH 7.5 37^o C)	10mM	1M	10µl	100µl
MgCl₂	10mM	1M	10µl	100µl
DTT	1mM	1M	1µl	10µl
H₂ O		mQ	79µl	790µl

	Conc.	Stock	100µl	1ml
Tris Cl (pH7.5 37^o C)	10mM	1M	10µl	100µl
NaCl	50mM	5M	10µl	100µl
MgCl₂	10mM	1M	10µl	100µl
DTT	1mM	1M	1µl	10µl
H₂ O		mQ	69µl	690µl

	Conc.	Stock	100µl	1ml
Tris Cl (pH7.5 37^o C)	50mM	1M	50µl	50µl
MgCl₂	10mM	1M	10µl	100µl
NaCl	100mM	5M	20µl	200µl
DTT	1mM	1M	1µl	10µl
H₂ 0		mQ	19µl	190µl

Denhardt solution 50x

(store at -20^o C)

	Conc.	Stock	50ml	300ml	1L
Ficoll 400	1%(w/v)	solid	0.5g	3.0g	10g
Polyvinilpyrolidone	1%(w/v)	solid	0.5g	3.0g	10g
BSA (fr V)	1%(w/v)	solid	0.5g	3.0g	10g
H₂ O		mQ	46.7ml	292.2ml	974ml

p=1.004

the solution should be filtered;
the solution could be prepared as 100x stock.

PBS (recipe 1)

pH(1x)=7.4, (store at NT or at 4^o C):

	1x	Conc.	Stock	100ml	500ml
KH₂ PO₄	1.7mM	17mM	136.1g/M	0.231g	1.157g
Na₂ HPO₄	5.2mM	52mM	142g/M	0.738g	3.692g
NaCl	150mM	1.5M	58.44g/M	8.77g	43.83g
H₂ O		mQ

	1x	Conc.	Stock	100ml	500ml
KH₂ PO₄	1.7mM	17mM	136.1g/M	0.231g	1.157g
Na₂ HPO₄ x7H₂ O	5.2mM	52mM	268.1g/M	1.394g	6.971g
NaCl	150mM	1.5M	58.44g/M	8.77g	43.83g
H₂ O		mQ

PBS (recipe 2) 10x

pH(1x)=7.3; (store at NT or at 4^o C)

	1x	10x	Stock	100ml	500ml
KH₂ PO₄	1.47mM	14.7mM	136.1g/M	0.2g	1.0g
Na₂ HPO₄ x7H₂ O	4.29mM	42.9mM	268.1g/M	1.15g	5.75g
NaCl	137mM	1.37M	58.44g/M	8.0g	40.0g
KCl	2.68mM	26.8mM	74.56g/M	0.2g	1.0g
H₂ O			mQ

PBS (recipe 3) 10x

pH(1x)=7.3; (store at NT or at 4^o C)

	1x	10x	Stock	100ml	500ml
KH₂ PO₄	2mM	20mM	136.1g/M	0.272g	1.36g
Na₂ HPO₄ x7H₂ O	10mM	100mM	268.1g/M	2.68g	13.41g
NaCl	137mM	1.37M	58.44g/M	8.0g	40.0g
KCl	2.7mM	27mM	74.56g/M	0.20g	1.01g
H₂ O			mQ

sterilize by autoclaving;
pH is concentration dependent; therefore check pH for diluted 1x buffer only;
pH of 10x should be ~6.8 (adjust by NaOH if necessary).

SSPE 20x

pH 7.4

	Conc.	Stock	1L
NaCl	3M	58.44g/M	175g
NaH₂ PO₄ xH₂ O	200mM	137.99g/M	27.6g
EDTA	20mM	372.24g/M	7.4g
H₂ O		mQ	926ml

adjust pH by 10N NaOH.

RNase A (DNase free)

solubilize 10mg/ml in 1-50mM AcONa (pH4.5);
incubate 15' in boiling water bath, then cool slowly to NT;
store at -20^o C, aliquote in use may be stored at 4^o C

RNAse storage buffer

(store at -20^o C).

	Conc.	Stock	1ml	5ml	10ml
Tris Cl, pH7.6	10mM	1M	10µl	50µl	100µl
Glycerol	50%	100%	0.5ml 0.630g	2.5ml 3.152g	5.0ml 6.305g
H₂ O		mQ	0.486g	2.43g	4.86g

Proteinase K

(store at +4^o C, long-term storage at �20^o C):
20mg/ml solution in 10mM Tris Cl, 1.0mM CaCl₂ , 30% glycerol.

EDTA/glycogen solution

20x (store at -20^o C):

	Conc.	Stock	1.0ml
EDTA	0.2M	0.5M	400µl
glycogen	1mg/ml	20mg/ml	50µl
H₂ O		mQ	550µl

DNase RQ1 buffer

10x (store at -20^o C):

	Conc.	Stock	1ml
Tris Cl (pH8.0₂₅ ^o _C )	0.4M	1M	400µl
NaCl	0.1M	5M	20µl
MgCl₂	60mM	1M	60µl
CaCl₂	0.1M	1M	100µl
H₂ 0		mQ	420µl

DNAse storage buffer

(store at -20^o C).

	Conc.	Stock	1ml	1.5ml	2.5ml	5ml	10ml
Tris Cl, pH 7.0	20mM	1M	20µl	30µl	50µl	100µl	200µl
NaCl	50mM	5M	10µl	15µl	25µl	50µl	100µl
DTT	1mM	1M	1µl	1.5µl	2.5µl	5.0µl	10µl
Glycerol	50%	100%	0.5ml 0.630g	0.75ml 0.9g	1.25ml 1.58g	2.5ml 3.15g	5.0ml 6.31g
H₂ O		mQ	0.485ml	0.73ml	1.21ml	2.42ml	4.85ml