Lab recipes gels 1% Agarose 80ml 1 100ml


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LAB RECIPES
GELS

1% Agarose - 80ml 1% -100ml

0.8g Agarose 1g

80ml 0.5X TBE 100ml

Add 2-3 μl of EtBr. Add 2-3 μl


0.7% Agarose - 80ml

0.56 Agarose

80ml 0.5X TBE

Add 2-3 μl of EtBr.



0.6% Agarose - 80ml

0.48g Agarose

80ml 0.5X TBE

Add 2-3 μl of EtBr.



2.5% MetaPhor - 80ml

2.0g MetaPhor

80ml 0.5X TBE(cool at -20 C for 10-20 min. before adding MetaPhor)

Add 2-3 μl of EtBr.


STOCK SOLUTIONS
0.5X TBE – 1 Liter

50ml 10X TBE

Fill to 1 L with dH20
10 X TBE– 1 Liter

800 ml dH20

121 g Tris (Trizma) Base

55 g Boric Acid

7.44 g EDTA

Mix well. Fill to 1 L with dH20. Mix well.


20% SDS

20g/100ml SDS

Filter Sterilize.

TE buffer 50 ml

10mM Tris 0.5 ml 1M Tris pH 8.0

1 mM EDTA 0.1 ml 0.5M EDTA pH 8.0

Super TE buffer 50ml

50 mM Tris 2.5ml 1M Tris

10 mM EDTA 1ml 0.5M EDTA

300 mM NaOAc(optional) 15ml 1M NaOAc

Fill to 50 ml
1M NaOH- 1 Liter

40g NaOH


Fill to 1 L with dH20.

1M KCI- 1 Liter(autoclaved)

74.56 g KCI

Fill to 1 L with dH20.

1M KOH- 1 Liter

56.1 g KOH

Fill to 1 L with dH20.
3M KOAc solution – 500 ml (autoclaved)

147g Potassium Asetate

142.5 ml Glacial Acetic Acid(CH3 COOH)

Bring volume to 500ml with ddH20. pH should be between 4.8 and 5.3.


10X PCR Buffer – 100ml

60ml dH20

10mM Tris 1.21 g Tris

2mM – 1.5mM MgCl2 0.305 g MgCl2

50mM 3.728 g KCl

Fill to 100 ml with dH20. Adjust pH to 8.3 at 20 C.

Autoclave.
100mM Glucose (Dextrose)

18.16 g/L of dH20 9.01 g Glucose

Add 50 ml dH20.

10XGel Loading Buffer “Type III”

0.42% Bromophenol Bule 20mg Bromophenol Bule

49.9% Glycerol in dH20 5ml Glycerol

5ml dH20

Store bulk stock in -20 C.

Store common use at 4C.



Stop Dye -1ml

100 μl 10X Loading Buffer

400 μl 0.5 M EDTA

100 μl 1.0% SDS

400 μl dH20

1kb Ladder – 50 ng/ μl

50.0 μl 1kb DNA (μg/μl)

840.0μl TE

10.0 μl (50mM) NaCl 100.0 μl Loading Buffer



X- Gal (20 mg/ml)

Stock solution is 20 mg/ml.

Use DMF (dimethyl-formamide) as solvent.

Add 40 μl fo 20 mg/ml stock to each plate.


IPTG

2 g IPTG


8 ml dH20

Dissolve. Bring volume to 10ml. Filter Sterilize.

Add 4 μl of stock to each plate.
TB Broth KH2PO4, K2HPO4

12 g Tryptone 2.31 g KH2PO4

24 g Yeast Extract 12.54 g K2HPO4

4 ml Glycerol 90 ml dH20

Fill to 900 ml with 900 ml dH20. Adjust volume to 100 ml.

Autoclave. Autoclave.

Add 100 ml of 0.17 M KH2PO4, 0.72 M K2HPO4.
ANTIBIOTICS
TABLE A.1 Antibiotic Solutions______________________________________

Working concentration

Stock solutionª stringent relaxed

concentration storage plasmids plasmids



Ampicillin 50 mg/ml in H²O -20ºC 20µg/ml 60µg/ml

Carbencillin 50 mg/ml in H²O -20ºC 20µg/ml 60µg/ml

Chloramphenicol 34 mg/ml in ethanol -20ºC 25µg/ml 170µg/ml

Kanamycin 10 mg/ml in H²O -20ºC 10µg/ml 50µg/ml

Streptomycin 10 mg/ml in H²O -20ºC 10µg/ml 50µg/ml

Tetracycline 5 mg/ml in ethanol -20ºC 10µg/ml 50µg/ml
ªStock solutions of antibiotics dissolved in H²O should be sterilized by filtration through a 0.22- micron filter. Antibiotics dissolved in ethanol need not be sterilized. Store solutions in light-tight containers.

bMagnesium ions are antagonists of tetracycline. Use media without magnesium salts (e.g., LB medium) for selection of bacteria resistant to tetracycline.
A.6 Bacterial Media, Antibiotics, and Bacterial Strains

Lab Stock Solutions (1 Liter):

5 M NaCl (autoclaved or not)

700 ml dH2O 1M NaOH 40g/l – 109%

Add NaCl 292.25 g 1M KOH 56.11- 14.0

Add dH2O up to 1liter
0.5 M EDTA pH 8 (autoclaved)

700 ml dH2O 20% SDS



Add Na2EDTA2H2O 186.1g

Add 10N Na OH until pH=8.0



Add dH2O up to 1liter
1M Tris 7.5 (autoclaved)

800 ml dH2O

Add Tris (Trizma) Base 121.1 g

Cool to room temp.

Add conc. HCI until pH=7.5(approx. 70 ml)

Add dH2O up to 1liter


1M Tris 8.0 (autoclaved)

800 ml dH2O

Add Tris (Trizma) Base 121.1 g

Cool to room temp.

Add conc. HCI until pH=8.0(approx. 42 ml)

Add dH2O up to 1liter


3M NaOAc pH 5.2 (autoclaved or not)

300 ml dH2O

Add NaC2H3O23H2O(NaOAc) 408 g

Add conc. Acetic acid until pH=5.2

Add dH2O up to 1liter
Lab Stock Solutions, cont. (volumes indicated):

Sterile dH2O (autoclaved)-pH 5.3

100 ml, 250 ml, 500 ml bottles

**Autoclave 20 min., slow exhaust (liquid)


Sterile 0.1% Phytager, 50ml (twise autoclaved)

0.05 g phytager

Add dH2O up to 50ml

Autoclave, slow exhause(liquid)

Mix several times while cooling to room temp

Wait one week

Autoclave,slow exhaust (liquid)

Mix several times while cooling to room temp


LB Liquid Medium (autoclaved)

800ml dH2O

Add Bacto tryptone 10g

Add Yeast Extract 5g

Add NaCL 5g

Mix well


Add dH2O up to 1liter

Mix well


Autoclaves 20min., slow exhaust (liquid)
10X TBE, 1liter (not autoclaved) pH=8.3

800ml dH2O

Add Tris (Trizma) Base 121.1 g

Add Boris Acid 55g

Add EDTA 7.44 g

Mix well


Add dH2O up to 1liter

Mix well
LB Plate (autoclaved)

800ml dH2O

Add Bacto trytone 10 g

Add Yeast Extract 5g

Add NaCL 5g

Add 17 g Bacto agar

Mix well


Add dH2O up to 1liter

Add 17 g Bacto agar

Mix well

Autoclaves 20 min., slow exhaust (liquid)

Allow to cool to 600C

Pour 15 mm plates, allow to cool


TE 50ml

10mM Tris 0.5ml 1M Tris pH 8.0

1mM EDTA 0.1ml 0.5M EDTA pH 8.0
Super TE 50ml

50mM Tris pH 7.5 2.5ml 1M Tris 7.5 stock

10mM EDTA pH 8.0 1ml 0.5M EDTA PH 8.0 stock

Bring volume to 50ml with dH2O


Extraction Buffer 100ml

200mM Tris pH 7.5 20ml 1M Tris pH 7.5

250mM NaCl 5ml 5M NaCl

25mM EDTA pH 8.0 5ml 0.5M EDTA

0.5% SDS pH 7.3-8.0(add after autoclave) 2.5ml 20% W/V SDS

Fill to 100ml with dH2O


Gel for Columns (for sequence) 100ml

Add 4g. biogel to 100ml 10mmM Tris, mix

Put for 12 hours, then discard Tris, add 100ml 10mM Tris, put for 12h, use
PSA

Add 50mg to 1ml dH2O, mix.



RNAase

Add 10mg to 1ml dH2O , boil in water(1000)

for 10 minutes. Store at -200C
Prepare 100ml 70% EtOH from 96% EtOH

X=70*100/96

X-volume of 96% EtOH
Acrylamide

Acr-29g


Bisacryl- 1g
CTAB precipitation solution 100ml

50mM Tris pH-8.0 1M Tris pH-8.0 5ml

10mM EDTA pH-8.0 0.5M EDTA pH-8.0 2ml

1% CTAB CTAB 1g

Autoclave
High salt TE 200ml

10mM Tris pH-8.0 1M Tris pH-8.0 2ml

0.1mM EDTA pH-8.0 0.5 M EDTA pH-8.0 40μl

1M NaCl 11.68g

Autoclave

2 x CTAB 300ml

100mM Tris pH-8.0 1MTris pH-8.0 30ml

20mM EDTA pH-8.0 0.5M EDTA pH-8.0 12ml

1.4M NaCl NaCl 24.5448 g

2% CTAB CTAB 6g

Autoclave



10 x CTAB(CTAB/NaCl) 100ml

0.7M NaCl NaCl 4.1g

10%CTAB CTAB 10g

Autoclave


Tris pH-8.0


To prepare 1 L of

1M Tris buffer at

pH


Combine stock solutions

Final ratio

(HCl:Base)



1M Tris HCl(ml)

1M Tris base (ml)

7.0

7.2


7.4

7.6


7.8

8.0



948.8

917.4


869.6

806.4


719.9

612.4


5.2

82.6


130.4

193.6


280.1

387.6


18.5

11.1


6.67

4.17


2.57

1.58


To 612.4ml 1M Tris HCl add ≈385ml 1MTris Base, (use 10M NaOH, HCl to correct pH)

Autoclave


Polyacrylamide gel





6%

7%

8%

10%

dH2O ml

24.1

23 98.5

21.7

19.4

10xTBE buffer ml

3.5

3.5 15

3.5

3.5

Polyacrylamide ml

7

8.1 34.7

9.3

11.6

TEMED μl

50

50 214.2

50

50

PSA μl

250

350 1000

350

350

Total volume ml

35

35 150

35

35



10 mM Tris pH-8.0 100ml

1 M Tris pH-8.0 1 ml

dH2O Fill to 100ml

Autoclave


Bromfenolsiniy
Saxaroza 4gr
BFS 25 mg
Tris assetat-EDTA 50X 200 mkl
disH2O 10 ml gacha
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