Dissolved Tris base and EDTA in 700mL distilled water, then added glacial acetic acid. Additional water was added to bring final volume up to 1L. Diluted to 1x for use.
50x M (100mL)
Na2HPO4
17.75g
KH2PO4
17g
NH4Cl
13.4g
Na2SO4
3.55g
Made up to 100mL with distilled water. Autoclaved for 15 minutes to sterilise.
ZY Media (400mL)
Tryptone
4g
Yeast Extract
2g
Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilise.
50x 5052 (100 mL)
Glycerol
25g
Glucose
2.5g
Alpha Lactose
10g
Dissolved glycerol in 40mL of distilled water. Dissolved glucose and alpha lactose in 40mL of distilled water. Combined two solutions, adjusted final volume to 100mL. Autoclaved for 15 minutes to sterilise.
TB Buffer (500 mL)
KCI(MW=74.5513)
9.3g
CaCl2.2H2O (MW=147.0146)
1g
PIPES (MW=302.4)
1.5g
MnCl2.4H2O
5.44g
Dissolved KCL, PIPES and CaCl2 in 300mL of Milli-Q water. pH adjusted to 6.7 using KOH. Dissolved 5.44g of MnCl2 in 100mL of Milli-Q water. Gradually added MnCl2 to the other solution. Final volume was adjusted to 500mL, filter sterilised with 0.22µm filter, then stored at 4°C
HEPES, pH 7.5 (200 mL)
HEPES
47.66g
KOH
As required
Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.
Tris-HCl, pH 8.0 (200ml)
Tris
24.228g
HCl
As required
Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.
1M Imidazole, pH 8.0 (400ml)
Imidazole
27.232g
HCl
As required
Dissolve imidazole in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 400mL.
1M EDTA, pH 8.0 (200ml)
EDTA
29.22g
NaOH
As required
Dissolved EDTA in 75% of total volume of Milli-Q water while adding NaOH to assist. Buffered pH to correct levels with NaOH, then adjusted final volume to 200mL.
Dithiotheritol 1M (5mL)
DTT
0.7715g
Dissolved DTT in Milli-Q water.
TE Buffer (100ml)
TRIS (1M) pH 8.0
1mL
EDTA(0.5M) pH 8.0
0.2mL
Combined reagents, adjusted final volume to 100mL with Milli-Q water.
Cas9 Buffer (100ml)
HEPES(1M) pH 7.5
2mL
KCl(1M)
15mL
Combined reagents, adjusted final volume to 100mL with Milli-Q water.
CaCl2 1M (100mL)
CaCl2
11.098g
KCl(1M)
15mL
Dissolved CaCl2 in 100mL of Milli-Q water.
Ammonium Bicarbonate 100mM (400mL)
Ammonium Bicarbonate
3.164g
Dissolved ammonium bicarbonate in 400mL of Milli-Q water.
Ammonium Bicarbonate 50mM (200mL)
Ammonium Bicarbonate
0.791g
Dissolved ammonium bicarbonate in 200mL of Milli-Q water.
Combined reagents and adjusted final volume to 400mL with Milli-Q water.
Iodoacetamide (DTT)
Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide to give a solution of 55 mM iodoacetamide - 15 mL of each is enough for up to 96 samples.
