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  Contact Information:

  3001 Mercer University Dr

  122 Duvall Bldg

  Atlanta, GA 30341

  (678)-547-6246

  moniri_nh@mercer.edu

   

Ampicillin (1000X)

 

IPTG (1000X)

 

Kanamycin (1000X)

 

G418

 

LB Agar

 

PBS (10X)

 

SDS-PAGE Running Buffer (10X)

 

SDS Sample Buffer (2X)

 

Glycerol Stock Preparation

 

TBST (10X)

Ig Stripping Buffer/ Protocol

Agarose Gel DNA Extraction

Agarose Gel Range

Wet IB Transfer Buffer (10X)

Semi-Dry IB Transfer Buffer (1X)

 TAE (10X)

RIPA Buffer (1X)

Krebs-Ringer Bicarbonate Buffer

HENS

Neutralization Buffer (for HENS)

 
   
   
   
   
   
   
     
 
 

 

 

Ampicillin (1000X, 100mg/ml)

1.0 g Ampicillin

Add dH2O to 10 ml

Sterile filter with 0.2um filter

Aliquot ~ 1ml, freeze @ -20°C

 

 

 

IPTG (1000X, 1M)

2.382 g IPTG

Add dH2O to 10 ml

Sterile filter with 0.2um filter

Aliquot ~ 1ml, freeze @ -20°C

 

 

 

Kanamycin (1000X, 50mg/ml)

682 mg kanamycin sulfate (Invitrogen stock pwd is 0.734 ug/mg pwd)

Add dH2O to 10 ml

Sterile filter with 0.2um filter

Aliquot ~ 1ml, freeze @ -20°C

 

 

 

G418 (100mg/ml stock)- use 1mg/ml final

In hood,

1.4 g G418 (di)sulfate powder (based on active weight, usually ~700ug active/mg powder)

10 ml sterile dH2O

Sterile filter with 0.2um filter into sterile tube

Use 1 mg/ml in media for selection

100 ul per 6 ml of media gives ~1.7mg/ml

Refresh with new media/G418 every 3 days

 

 

LB Agar

16.0 g LB

7.5 g Agar

500 ml dH2O

Autoclave, cool

Add antibiotic, pour

Store at 4°C

 

 

 

PBS (10X) (FINAL CONC IN PARENTHESIS)

80.0 g NaCl  (1.37 M)

2.0 g KCl     (26.8 mM)

14.2 g Na2HPO or 27.0 g Na2HPO4*7H2O  (0.1 M)

2.4 g KH2PO4 (17.6 mM)

dH2O to 800 ml stirring in beaker

Adjust pH to 7.4

Adjust to 1 L

Autoclave

 

 

 

10X SDS-PAGE Running Buffer-Denaturing

29 g Tris Base

144 g Glycine

10 g SDS or 50 ml of a 20% SDS solution

dH2O to 1L

Do NOT adjust pH

 

For Non-Denaturing, same as above w/out SDS

 

 

 

2X SDS-Sample Buffer

25 ml 0.5M Tris-HCL, pH 6.8

20 ml glycerol

20 ml 20% SDS

5 ml 0.1% BPhB

dH2O to 100 mL

 

 

 

 

Agarose Gel Separation Range

Agarose %                 Range of Separation

     0.3                                   60 - 5 kb

     0.6                                   20 - 1 kb

     0.7                                   10 - 0.8 kb

     0.9                                     7 - 0.5 kb

     1.2                                     6 - 0.4 kb

     1.5                                     4 - 0.2 kb

     2.0                                     3 - 0.1 kb

 

 

 

Glycerol Bacterial Stock

1500 ul bacterial broth

500 ul sterile 60% glycerol

Label, freeze at -80°C

 

 

 

 

TBST (10X)

30 g Tris Base

88 g NaCl

2 g KCl

970 mL dH2O

Bring pH to 7.5

5 ml Tween-20

Adjust to 1 L

 

TBS (10X)

30 g Tris Base

88 g NaCl

2 g KCl

970 mL dH2O

Bring pH to 7.5

Adjust to 1 L

 

 

 

Immunoglobulin Stripping Buffer & Protocol

0.75 ml b-ME

10 ml of 20% SDS

0.76 g Tris Base (62.5mM final) OR 12.5 ml  0.5 M Tris-HCl pH6.8

dH2O to 100 mL

 

Protocol

1)  Add membranes and buffer to small plastic box with tight fitting lid

2)  Incubate 50°C for 45 minutes

3)  Rinse membrane with dH2O

4)  Wash 3x in TBST, 5 min each

5)  Take on to blocking step

 

 

Glycine Stripping

Dissolve 188mg glycine in 75 ml dH2O

Adjust pH to 2 with HCl

Add 5ml 20% SDS (1% final)

QS to 100 ml with dH2O

 

Protocol

1)  Warm buffer to 50°C for 15-20 min

2)  Soak blot in buffer in container with lid at RT for 30 min

3)  Rinse blot with dH2O 4-5 times

4)  Wash 3 times with TBST ~5 min each

5)  Take on to blocking step

 

 

 

 

 

Wet Immunoblot Transfer Buffer (10X)

30.25 g Tris Base

144 g Glycine

 

Add 0.1% SDS only if protein of interest >100KDa

 

For 1X, dilute to by:

100 ml 10X Transfer Buffer

200 ml MeOH

700 ml dH2O

 

 

 

Semi-Dry Immunoblot Transfer Buffer (1X)

5.82 g Tris Base  (48mM)

2.93 g Glycine  (39mM)

Dissolve in 700 ml dH2O

Add 200 ml MeOH

Add 3.75 ml of 10% SDS (or 1.875 ml of 20% or 0.0375 g SDS)   (0.0375%)

Adjust to 1 L

pH should be ~9.2, do NOT adjust

 

 

 

TAE (10X)

48.4 g Tris Base

11.42 ml Glacial Acetic Acid

20 ml 0.5 M EDTA (pH 8)

dH2O to 1 L

 

 

 

 RIPA Buffer (1X) (Denaturing)

25 mM Tris-HCl (pH 7.6)

150 mM NaCl

1% NP40

1% Na Deoxycholate

0.1% SDS

 

-  Add 12.5 ml of 1 M Tris-HCl

-  Add 15 ml of 5 M NaCl

-  Add 5 ml of NP40

-  Add 50 ml of 10% NaDeoxycholate stock, or add 5 g of NaDeoxycholate powder

-  Add 2.5 ml of 20% SDS or add 0.5 g of SDS powder

-  dH2O to 500 ml

 

For non-denaturing (e.g., Co-IP), remove SDS and Na Deoxycholate, replace NP40 above with 0.5% final concentration.

Krebs-Ringer Bicarbonate Buffer
1.5mM MgSO4
4.7mM KCl
1.3 mM CaCl2
1.2mM KH2PO4
25mM NaHCO3
11.7mM Glucose
118mM NaCl

pH Adjusted on day of use with O2/CO2 Gas

- 369.7mgMgSO4
-350 mg  KCl
-191mg CaCl2
-163.3mg KH2PO4
-2.1g NaHCO3
-2.108g Glucose
-6.895g NaCl
-1000ml diH2O

 

HENS
25mM HEPES pH 7.5
1mM EDTA
1% SDS
10µM Neocuproine

-Add 3.25g of HEPES
-186mg EDTA
-1mg Neocuproine
-5g SDS
-450 ml diH2O
-Adjust pH to 7.5
-qs to 500ml

For HENS10 dilute HENS buffer 1:10 in diH2O

 

Neutralization Buffer (for HENS protocol)
20mM HEPES pH 7.5
1mM EDTA
100mM NaCl
0.5% Triton

-Add 2.6g HEPES
-186mg EDTA
-2.922g NaCl
-450ml diH2O
-Adjust pH to 7.5
-Add 2.5 ml Triton-X 100
-qs to 500ml

 

 

 

 
     
     
                                                                                                                                                                      
Last Updated: 2/3/2019
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