Reaction mixtures, Media and Solutions:

Reaction Mixtures, Media and Solutions:

These are used by me in my work;

5� End labeling:

Use T4 Polynucleotide kinase.

10 x buffers:

0.5M Tris-Cl pH 8,

0.1 M MgCl2,

0.1M DTT,

3� Recessive end labeling:

One can use Klenow fragment, T4 DNA-pol or T7 DNA-pol,

Use 10x Klenow Buffer: containing 10x Hepes buffer pH 6.6.

0.5 M Tris-Cl pH 7.6,

0.1 M Mg Cl2,

RNA labeling:

10x Transcription buffer:

0.4 M Tris-Cl pH 8,

0.08 M Mg Cl2,

0.5 M NaCl,

0.02 M Spermidine-HCl.

5� end labeling:

10x buffer:

0.5 Tris-Cl pH 8,

0.1 M Mgcl2,

0.1 M DTT (Dithiothretol).

Use T4 polynucleotide kinase.

Labeling 3� overhangs:

10x buffer:

1M Potassium cocodylate pH 7.2,

20mM cacl2,

2mM DTT.

Use Terminal Transferase.

TBE buffer:

10x buffer:

108gms Tris base,

55gms Borate,

40ml 0.5M EDTA (pH 8.0).

TAE buffer: 50x:

242 gm Tris base,

57.1 ml Glacial acetic acid,

100ml 0.5M EDTA ph 8.0.

Strength of the Agarose gel for DNA fragments:

0.3% 5-50KB,

0.5% 1-25KBP,

0.8% 700bp- 10KBP,

1% 500bp � 10KBP,

1.5% 200bp- 5KBP,

2% 100bp-2KBP,

2.5% 20bp-1KBP.

20x buffer SSC (NaCl-Na citrate):

174.3 gms NaCl,

88.2 Na citrate,

Make it to 1000ml, pH 8.0.

Hybridization buffer 10X:

0.5M Sodium phosphate (pH 7.2),

7% SDFS,

1mM EDTA,

50% Farmamide (ultrapure0,

1x Denhardt sol.

100ug/ml Calf thymus sheared DNA.

Wash buffer:

0. X ssc+0.1%SDS at room Tm,

0.2x SSC +0.1 % SDS at room Tm,

0.2x SSC + 0.1% SDS at 62^o C.

Northern Blotting:

Denaturation buffer:

DMSO4- 20ul,

0.1M Na H2 PO4 pH7- 4ul,

Deionized glyoxal- 5.9ul,

RNA -10ug.

Mix and incubate at 50^0C for 60 minutes,

Then mix with glyoxal loading buffer;

50% glycerol,

10mM Na H2Po4 pH 7.0

0.25% Xylene cyanol,

0.25% Bromophenol blue.

Glyoxal removal buffer:

Submerge the membrane in 20mM Tris-Cl pH 8.0 at 65 ^oC for few minutes.

Preparation of Gel for northern blotting:

RNase is the most dreaded enzyme in all labs.� Take precautions to avoid the contamination with RNase.

10mM NaH2PO4 ph 7.01 gm of Agarose,

All RNase free.

Running buffer is the same.

Gel loading buffer:

0.25% Bromophenol blue,

0.25% Xylene cyanol,

40%sucrose or

15% Ficol in water.

Denhardt solution 100 x:

10gm Ficol 400,

10gm PVP,

10gm BSA�

Water to 500ml.

Phage 6M buffer:

50mM Tris-Cl pH 7.8,

8mM Mgcl2,

100mM NaCl,

0.1% Gelatin.

Phage diluting buffer:

10mM Tris-Cl pH 7.5,

10mM MgCl2.

Stock EtBr:

10mg/ml.

Depurination solution:

0.25M HCl.

Denaturation buffer:

1.5M NaCl,

0.5M NaOH.

�Neutralization buffer:

1M Ammonium acetate,

20mM NaOH,

Tris Cl and NaCl.

SDS PAGE buffers:

Stock: 29.2 gms acrylamide,

0.8 gms Bis acrylamide,

Make it to 100ml, store it in cold room.

Separation Gel:

Tris-Cl 10ml (1.5M Tris),

SDS 0.4ml (10% SDS),

Ammonium per sulfate 0.170ml,

TEMED 0.032ml.

Add 16ml of Acrylamide stock solution,

Make it to 40ml.

This is for 10 % gel.

Stacking gel:

Stock acrylamide 1.7ml,

Triscl-6.8 1.25ml (1 M),

SDS 0.1ml,

APS 0.075 ml (10%stck),

TEMED 0.016ml.

Make it to 10ml.

4x sample buffer 10ml:

SDS 2ml,

Tris pH 6.8, 1.2ml (1M Tris),

Glycerol 4ml,

Bromophenol blue 2.5mg,

Coomassie Brilliant Blue 2.5 mg.

B-Mercaptoethanol 2ul/20ul.

Gel Running Buffer:

Tris 15gm,

SDS 5gms,

Glycine 90gms,

Make it to 1liter.

Gel staining soln.:

Methanol 400ml,

Acetic acid 100ml,

Water 500ml,

Coomassie Blue 200 mg/200ml of the above solution.

Destaining solution: same as the above with out the stain.

Sequencing Gel:

TBE buffer (5x):

Tris base 54gms,

Boric acid 27.5 gms,

EDTA 20ml (0.5 M pH 8.0).

Gel loading buffer:

Bromophenol blue 0.25%,

Xylene cyanol 0.25%,

Sucrose 40% or Ficol 15%

Prepare 5% Polyacrylamide-Urea Gel;

Acrylamide stock 30%,

Urea (7M stock),

TBE (10x) - 5ml,

Acrylamide 30%- 25ml,

Urea -21.gms,

APS- 200ul,

TEMED- 25ul

Western Blot Buffers:

Transfer buffer:

Tris-Glycine SDS containing 20% methanol.

Membrane treatment Buffer:

TBS (50mM Tris *.0, 150mM NaCl) or PBS,

TBST or PBST,

PB=phosphate buffer, (10mM NaPHO4 (7.0) + 150mM NaCl)

TB=Tris buffer,

T= Tween (.03%),

Blocking buffer: TBST + Skimmed milk 0.5 to 3%; or 0.1%BSA

Washing 2 to 3 times.

Incubation with IgG in PBS (T) or TBS (T) buffer;

Incubation with anti-antibody conjugated with AP in PBS/TBS,

Washing twice to three times;

Alkaline phosphate buffer (pH9.5); 5mM Mgcl2, 50mM Na2CO3 (or 100mM NaCl, 100mM Tris-Cl pH 9.5, 5mM MgCl2.

Then add Nitro BlueTetrazolium (NBT) (100ug/ml, and 5, Bromo 5-chloro 3-Indolyl phosphate (BCIP) 50ug/ml. ��

Culture media:

Bacterial culture media: LB medium (Luria Broth):

10 gms Bacto Tryptone,

5 gms Bacto-yeast extract,

10 gms NaCl,

Make it to 1 liter.

Phage buffer:

200mM Tris-Cl 7.4,

100mM NaCl,

10mM MgSO4.

Supplement medium:

�For Y1090 cells:

10gm Bacto-Tryptone,

5gm Bacto-yeast extract,

5gm NaCl,

pH 7.5;

50ml LB

0.5 ml 20% maltose,

0.5ml of 1M MgSO4.

TEP buffer:

100mM Tris-Cl 7.4,

10mM EDTA,

1mM PMSF (10mM PMSF in EtOH),

SM buffer:

50mM Tris-Cl 7.5,

100mM NaCl,

8mM MgSO4,

0.01% Gelatin.

PCR buffer:

670mM Tris-Cl (.0,

67mM MgCl2,

1.7mg/ml BSA, 160mM (NH) 4 SO4.

Yeast culture media:

YP:

10gm Yeast extract,

20gms Bacto-peptone,

Make it to 1 liter.

YPD:

YP + 0.1 volumes of 20% Glucose.

LB for Bacterial transformation or electroporation:

Bacto-Tryptone 2%,

Bacto-yeast extract 0.5%,

NaCl 10mM,

KCl 2.5mM;

Mgcl2+MgSO4- 22mM,

PH 6.5;

Mg and Mg So4 -20mM each,

TFB:

K MES 10mM,

KCl 100mM,

MnCl2.4h2O- 45mM,

CaCl2-2H2O- 10mM,

H4COCl3 (Cobalt chloride)-3mM,

pH- 6.2.

FSB:

K.Acetate 10mM,

Glycerol 10%,

KCl 100mM,

MnCl2 45mM,

CaCl2 10mM,

HACoCl3 3mM. Adjust the pH to 6.2.

��

Random primining:

10X Hepes pH 6.6

Klenow

3 dNTPs

ααα32P CTP ( high Ci/mM).

RNA Labeling:

Stock:

10x Transcription buffer,

0.4 Tris-Cl pH 8,

0.8M MgCl2,

0.5M NaCl,

0.2M Spermidine,

RNA polymerase,

α32P-UTP

Purify with G-50 column

Random primining:

10X Hepes pH 6.6

Klenow

3 dNTPs

alpa 32P CTP ( high Ci/mM).

RNA Labeling:

Stock:

10x Transcription buffer,

0.4 Tris-Cl pH 8,

0.8M MgCl2,

0.5M NaCl,

0.2M Spermidine,

RNA polymerase,

32P-UTP

Purify with G-50 column

Meanwhile one has to have freshly grown E.coli strains such as Y1089 (lysogenic) or Y1090 (lytic).� These have to be grown in LB medium containing MgSO4 (20mM) and maltose 2%.� The density of the cells to be at 0.5 to 0.7 OD.

LB medium:

10 gm Bacto Tryptone,

5 gm Bacto yeast extract,

2% maltose,

20mM MgSO4,

Adjust the pH to 7.5.

Phage buffer:

200 mM Tris-Cl 7.4,

100 mM NaCl,

10 mM MgSO4

A list of Solutions and Buffers is unending- the above are some basic solutions used by me.