Media
LB broth (1L)
Tryptone: 10g
Yeast Extract: 10g
NaCl: 5g
Dissolved in 1L MQ and pH adjusted to 7.0.
LB Agar(1L)
Tryptone: 10g
Yeast Extract: 10g
NaCl: 5g
Agar: 15g
Dissolved in 1L MQ and pH adjusted to 7.0
Antibiotic Stocks
Carbenicillin
50mg/ml in MQ. Stored at -20oC.
Kanamycin
50mg/ml in MQ. Stored at -20oC.
Buffers for mini prep
0.5M tris pH 8:
6.055g Tris
Dissolved in 80ml MQ. pH adjusted to 8 with0.1M HCl (~20ml). Volume was made up to 00ml with MQ. Autoclaved and stored at RT.
0.5M NaCl:
NaCl: 2.922g
Dissolved in 100 ml MQ. Filter sterilized and stored at room temperature.
100mM EDTA:
EDTA: 2.922g
Dissolved in 80 ml MQ and pH adjusted to 8 with NaOH(~12ml). Volume made up to 100 ml with MQ. Autoclaved and stored at RT.
RNase A (10mg/ml)
0.5M Tris pH 8:1ml
0.5 M NaCl: 1.5ml
MQ: 25ml
pH adjusted to 7.5 with 0.1 M HCl and volume made up to 50 ml with MQ. Autoclaved and cooled.
0.02g RNase A was dissolved in 2ml of 10mM Tris pH 8 and 15mM NaCl buffer. Aliquoted into two microfuge tubes 1ml each and heated to 100oC for exactly 15min. Cooled at room temperature and stored at -20oC.
P1 Buffer (resuspension buffer) pH 8
TrisCl: 50 mM
EDTA: 10 mM
RNaseA: 100 µg/ml
pH adjusted to 8 with HCl
6.06g Tris Base dissolved in 800 ml of MQ, followed by 3.75g of EDTA.2H2O and dissolved. pH was adjusted to 8 with HCl and volume made up to 1L with MQ. Autoclaved and cooled. 10 ml RNaseA (from 10mg/ml stock) was added and stored at 4oC. (10ml RNase A from above stock solution was added to 1L P1 buffer)
P2 Buffer (lysis buffer):
NaOH: 200mM
SDS: 1% (w/v)
2.4g of NaOH pellets were dissolved in 200ml of autoclaved MQ, followed by 3g SDS. Volume was made up to 300ml and stored at RT.
P3 Buffer (neutralization buffer) pH 5.5:
Potassium acetate: 3M
88.35g potassium acetate was dissolved in 150 ml of MQ. pH adjusted to 5.5 with glacial acetic acid (~100ml). Volume was made up to 300ml with MQ and stored at RT.
Solutions for Agarose Gel Electrophoresis
TE
Tris.Cl: 10mM
EDTA: 1mM
pH adjusted to 8 with HCl. Autoclaved and stored at room temperature.
6X Agarose gel loading Dye
Sucrose: 4g
Bromophenol Blue: 2.5mg
Both components were dissolved in 6ml TE buffer. Volume made up to 10 ml with TE buffer and stored at RT.
Ethidium Bromide (10mg/ml)
Ethidium Bromide: 1g
Dissolved in 100ml autoclaved MQ and stored in amber colored bottle or wrapped with aluminium foil at 4˚C.
50X TAE
Tris base: 242g
Na2EDTA.2H2O: 37.2g
Dissolved in 900ml MQ followed by 57.1ml glacial acetic acid and volume made up to 1L.
DNA ladder stock 1kb/100bp/50bp:
NEB DNA ladder: 10µl
6X Agarose Gel Loading Dye: 10µl
90µl of autoclaved MQ was added to make volume up to 100µl and stored at -20oC.
Solutions for blue/white selection of recombinants
X-gal
X-gal: 20mg
Dissolved in 1ml dimethylformamide (DMFA). Stored at -20oC in amber colored eppendorf or wrapped in aluminium foil.
IPTG
IPTG: 0.4g
Dissolved in 1ml MQ and volume made up to 2ml with MQ. Filter sterilized and dispensed into 1ml aliquots and stored at -20oC.
LCIX plates
250µl of carbenicillin (stock 50mg/ml) was added to 250ml of lukewarm LB agar to get working concentration of 50µg/ml. The plates were poured and allowed to solidify. Stored at 4oC.
Solutions for SDS-PAGE
30% Acrylamide mix
Acrylmamide: 29g
N,N- methylene bis acrylamide:1g
MQ: 100ml
N.N-methylene bis-acrylamide dissolved in 50 ml MQ followed by acrylamide. Volume made up to 100ml with MQ and stored at RT in amber colored bottle.
1X Tris Glycine:
Tris base: 3.02g
Glycine: 18.8g
Dissolved in 900ml MQ and added 10 ml of 10% (w/v) SDS. Volume made up to 1L and stored at RT.
1M Tris pH 6.8:
Tris base: 12.11g
Dissolved in 70ml MQ and pH adjusted to 6.8 with conc. HCl. Volume made up to 100 ml and stored at RT.
1.5M Tris pH 8.8:
Tris base: 45.417g
Dissolved in 200 ml MQ and pH adjusted to 8.8 with conc. HCl. Volume made up to 250 ml and stored at RT.
10% SDS solution:
5g SDS dissolved in 50 ml MQ and stored at RT.
10% Ammonium Persulphate (APS):
1gm APS dissolved in 10 ml MQ and stored at 4˚C.
TEMED(N,N,N′,N′tetramethylethylenediamine)
4X SDS Gel Loading Buffer:
1M Tris pH 6.8: 2.5ml
β- mercaptoethanol: 2ml
SDS: 0.8g
Bromophenol Blue: 1.7mg
Glycerol: 4ml
Liquoted in microfuge tubes and stored at -20˚C.
Coomassie Brilliant Blue Stain:
Coomassie Brilliant Blue R250/G250: 1.25g
Methanol: 450 ml
MQ: 450 ml
Glacial acetic acid: 100ml
1.25g Coomassie Brilliant Blue R250/G250 dissolved in 900 ml methanol:water(1:1w/v) and added 100ml of glacial acetic acid. Solution filtered through Whatman no.1 filter paper to remove any particulate matter and stored at RT.
Destain:
Methanol: 450ml
MQ: 450ml
Glacial acetic acid: 100ml
Mixed and stored at RT.
Casting 12% SDS-PAGE gel
Resolving gel (volume for two gels):
MQ: 3.4 ml
30% acrylamide: 4ml
1.5M Tris pH 8.8: 2.5ml
10% SDS: 0.1ml
10% APS: 50µl
TEMED: 6µl
Stacking gel (volume for two gels):
MQ: 2.7ml
30% acrylamide: 0.67ml
1.5M Tris pH 6.8: 0.5ml
10% SDS: 40µl
10% APS: 40µl
TEMED: 5µl
Solutions for Western Blotting
Lysis buffer:
NP40 lysis buffer (Invitrogen): 900µl (stored at -20˚C)
Protease inhibitor: 1 tablet of protease inhibitor dissolved in 1ml of AMQ to make 10X protease inhibitor.
100µl of 10X protease inhibitor was added to 900ml of NP40 lysis buffer and stored at -20˚C.
TBST(Tris Buffer Saline Tween):
NaCl: 9g
1.5M Tris pH 8.8: 5ml
1M Tris pH 6.8: 5ml
Dissolved in 800ml MQ and added Tween 1ml. Volume made up to 1L with MQ.
1X Transfer Buffer:
Tris base: 3.03g
Glycine: 14.41g
Dissolved in 800ml MQ and 200ml methanol. Stored at 4˚C.
5% blocking Solution:
2.5g of Skimmed Milk or BSA dissolved in 50 ml of TBST and stored at 4˚C.
Stripping Buffer:
SDS: 2g
14.1M β-mercaptoethanol: 0.71ml
1M Tris HCl pH 6.7: 6.25ml
Dissolved in 80 ml. Volume made up to 100ml and stored at RT.
Solutions for Protein expression and purification
1M IPTG
0.4g IPTG was dissolved in 1ml MQ and volume made up to 2ml with MQ. Filter sterilized and dispensed in 1ml aliquots. Stored at -20oC.
1M Imidazole
Imidazole: 6.808g
Imidazole was dissolved in 100ml MQ and stored at 4oC.
1M Tris (pH 8.0)
Tris base: 12.11g
Dissolved in 80ml MQ and pH adjusted to 8.0 with 0.1M HCl. Volume was mad up to 100ml with MQ and stored at RT.
Loading Buffer
50mM Tris pH 8.0
150mM NaCl
10% Glycerol
Wash Buffer
50mM Tris pH 8.0
10% Glycerol
Elution Buffers
50mM Tris pH 8.0
10% Glycerol
Varying concentrations of Imidazole: 10mM, 50mM, 100mM, 200mM and 300mM.