ETHIDIUM BROMIDE (EtBr 10mg/ml) RECIPE (5,000x)

EtBr 0.1g

ddH2O to 10 ml

*heat up to 50 deg to get into solution

LBA media/agar

LB premix (difco) 25g

agar 20g

water to 1L

-autoclave for 20 minutes

-cool to 50C (cool enough to touch while still a liquid)

- add 1ml of 100 mg/ml ampicillin stock

-mix well by inversion

- add 20ml LBAgar+amp per 100mm sterile peti dish

1 % AGAROSE GEL RECIPE (300 ml)

Agarose 3.0 g

0.5X TBE 300 ml

-microwave until in solution

-cool until 60 deg ( barely hold in hands)

-add EtBr

EtBr (20,000x) 15ul

Because we are adding the EtBr and then allowing the gel to sit at RT and under light,

we must add 0.5x the amount of EtBr (2ug/ml). Therefore it is a 5,000x solution.

TBE 5X RECIPE (1L)

Trizma base 54 g

Disodium EDTA 3.75 g

Boric Acid 27.5 g

ddH20 to 1.0 L

Dilute 1 in 10 with ddH20. The PH of the 1X buffer should be 8.3. DO NOT ADJUST THE PH.

* Trizuma base is tris base; tris that is not PH and is basic.

* Trizuma hydrocholride has been nuetralised to ph 7.

Tris-acetate-EDTA (TAE) 50 X RECIPE (1L)

To 900 ml of ddH20 add:

Tris base 242 g

Glacial acetic acid 57.1 ml

disodium EDTA (fw: 372.2g) 18.6 g

ddH20 to 1.9 L

*Glacial is the unadulterated or pure powder/solution

*EDTA only goes into solution at PH 8.0

PBS 10X (1L)

dissolve the following into 800 ml ddH2o

NaCl 80 g

KCl 2 g

Na2HPO4 14.4 g

KH2PO4 2.4 g

--> adjust to PH 7.4

--> adjust volume to 1L with ddH20

--> autoclave

can use henderson-hasselhoff equation to calculate the amount of monobasic and dibasic

phosphate to acheieve exactly 7.4 Ph

SOB MEDIA (1L) "super optimal culture"

Add to 900ml ddh20

Bacto tryptone 20g or SOC + 20ml of 1M filtered glucose solution

Bacto Yeast extract 5g ( add after autoclaving)

5M NaCl 2ml

1M KCl 2.5ml

1M MgSo4 20ml

Ph to 7.5 with 1M NaOH (~25ml)

For 1 L of LB, mix the following ingredients in a 2L glass container with a stir bar until clear.

10 g Bacto-tryptone

5 g Bacto-Yeast Extract

5 g NaCl (sodium chloride)

add water to 1 L

Add 200µL 5N NaOH (or 400µL 2.5M NaOH) to PH 7.5 --> for 500ml add 165ul of 10M NaOH (9/26/06)

Aliquot as desired – screw on top or cover with aluminum foil, send out to be autoclaved.

10X(6x) ORANGE G DNA LOADING BUFFER

To 35 ml of ddH20 (TE better, no growth) add in the following order:

100mg of Orange G powder

15ml of glycerol

- bad do not use as 6x, may use as 10X

EASY GREEN LOADING DYE (10X) - wendy chao.com

For 10ml of loading dye

* 2.5 g Ficoll-400

* 1 ml 1 M Tris-Cl, pH 7.4

* 2 ml 0.5 M EDTA

* Bring to 10 ml with ddH2O, heating to 65°C to dissolve.

* Add 25-50 mg each xylene cyanol and Orange G.

If needed, adjust dye intensity with colorless 10x buffer. Store at room temperature.

- for 0.25mg/ml dye (each) 2ul max to sample. Excess causes DNA distortion.

TE BUFFER (10X) (100ml)

Tris-EDTA ratio is 10:1

to 85 ml ddH20 add:

10ml 1M Tris-HCl (pH 8)

400ul 0.25M EDTA

Lambda DNA digested with bstE II mixed with loading dye (DNA ladder) -> 50ng/ul

10ul Lambda DNA (500ug/ml)

15ul 10X orange loading dye (really 6x)

75ul ddh20

Phi 174 DNA digested with hae III with loading dye (DNA ladder)

5ul Phi 174 (1000ug/ml)

15ul 10X orange loading dye (really 6x)

80ul ddH20

2-Log DNA ladder with loading dye (100ng/ul)

10 ul 2-log DNA (1ug/ul)

15ul 10X orange loading dye

75ul water

STET (500ml) [boiling mini-preps]

5ml 1M Tris Ph8

10ml 5M NaCl

1ml 0.5 EDTA ph8

25ml triton X-100 (ADD LAST beacause highly viscuous)

-> add water to 500ml

-> Check that PH is 8.0

LYSOZYME

10mg/ml

RNAase A

100ug/ml

resuspend in 10mM tris 7.5

N2/B27 - CDM media for feeder layer free ESC culturing (50ml)

23.25 ml DMEM

23.25 ml Ham's F12

1 ml B27 sup. (50X)

0.5 ml N2 supp

1 ml L-glutamine (0.1M - 50X)

11 ul B-Mecaptoethanol (0.5M B-Me)

0.5 ml non-essential AA (10mM, 100X)

0.5 ml BSA (50mg/ml - 100X)

->add 10 ul bFGF (100ng/ul)

Does not need to be Phed(~7.1) is slightly yellow

BG-K media (50ml)

41.5 ml DMEM/F12

7.5 ml KOSR

500 ul NEAA

1 ml glutamine (0.1M)

0.5 ml embryo-max 2-mercaptoethanol (100X)

--> add

does not need to be Phed (~7.18) is yellow

10X NEB T4 DNA ligase buffer (1ml)

50ul 1M Tris 7.5 (20X)

100ul 1M MgCl2 ( 10X)

100ul 1M DTT ( 10X)

100ul 100mM ATP (10X)

25ul 10 mg/ml BSA ( 40X)

625ul H20

3M Potassium Acetate PH 4.8 (500ml)

147.2g KAc

--> fill to 300ml with h20

add 210ml of glacial acetic acid