Solutions and Media
Antibiotic Solutions
Should be kept in the -4 degrees fridge
Ampicillin
Stock: 50 mg/ml
To make 10 ml, add 0.5g to 10 ml dH2O
Tetra
Stock: 10 mg/ml
To make 5 ml, add 0.05 g Tet to 2.5 ml 100% EtOH, 2.5 ml dH2O (cover with foil)
Strep
Stock: 10 mg/ml
To make 10 ml, add 100 mg of Strep to 10 ml dH2O
Kanamycin
Stock: 50 mg/ml
To make 10 ml, add 0.5 g of Kan to 10 ml dH2O
BE SURE TO FILTER STERILIZE AMP AND STREP WITH A 0.22-MICRON FILTER!!!
For Plates
For Amp Plates: Add 3 ml Amp per Liter of LB
For Amp/Tet plates: Add 1.25 ml Tet and 2 ml Amp per Liter of LB
For Strep Plates: Add 5 ml of Strep to 1 Liter of LB
For Strep/Tet plates: Add 750 ul of Tet and 2.5 ml of Strep to 500 ml of LB
For Kan plates: Add 1 ml of Kan to 1 Liter of LB
BE SURE THE LB IS COOLED TO AT LEAST 120 DEGREES F BEFORE ADDING ANTIBIOTICS!!!
Ampicillin Plates
For 1 Liter:
1 L LB
16 g bacto-agar
1. Autoclave.
2. Add 2 ml of 50 mg/ml ampicillin to cooled (50 degrees C or 120 degrees F) LB/agar.
3. Pour plates.
4. Allow them to sit overnight.
Store in cold room.
Ampicillin/XGAL/IPTG plates
For 250 ml:
250 ml LB
4 g bacto-agar
1. Autoclave.
2. Add 0.3 ml ampicillin (50 mg/ml), 5.95 ml IPTG (200 mg/ml), and 1 ml XGAL (20 mg/ml) to cooled (but not solid) LB/agar (normally around 50 degrees C or 120 degrees F)
3. Pour plates.
10X Electrode Buffer (aka Tris-Glycine)
For 1 Liter:
144 g glycine
30 g tris(Trizma Base)
1. Heat to get into solution.
2. Filter with filter paper.
0.5 M EDTA
100 ml 0.5 M EDTA pH8
186.1 g EDTA/L H2O
pH to 8.0 to get into solution, can heat gently to help solubility.
Gel Denaturing Solution
For 1 Liter:
58.5g 1 M NaCl
22 g 0.5 N NaOH
Gel Destain
For 1 Liter:
400 ml methanol
100 ml concentrated acetic acid
500 ml distilled water
Gel Neutralizing Solution
For 1 Liter:
121.1 g of 1.0 M Tris
87.67 g of 1.5 M NaCl
30 mL of HCl
1 L of dH2O
pH: 8.0
Gel Stain
For 1 Liter:
2 g Coomassie Blue R250
250 ml methanol
100 ml concentrated acetic acid
1. Dissolve coomassie blue in the methanol.
2. Wait 1 hour.
3. Add concentrated acetic acid.
4. Wait 1 hour.
5. Filter through filter paper.
Imidazole
For 1 Liter:
102.12 g imidazole
1 l of H2O
pH to 7.3 (use concentrated HCl)
LB
For 1 Liter:
10 g sodium chloride (NaCl)
10 g tryptone
5 g yeast extract
pH to 7.2
Needs to be autoclaved the same day it is made.
Nytran Hybridization Buffer
For 1 Liter:
300 ml 20X SSPE (pH: 7.7)
50 ml 20% SDS
5 ml sss DNA (10 mg/ml)
500 ml deionized formamide
145 ml ddH2O
pH: 7.4
Filter Sterilize
Aliquot into 50 ml tubes, freeze at -20 degrees C
Nytran Prehybridization Buffer
For 200 ml:
60 ml 20 X SSPE
40 ml 50 X Denhardt's
10 ml 20% SDS
1.0 ml sss DNA (10 mg/ml)
89 ml H2O
1. Mix and heat
2. Filter Sterilize (if possible)
3. Aliquot into 50 ml tubes and freeze at -2 degrees C
4. Label "NPH" and date
NZM Medium
For 1 Liter:
950 ml of deionized H2O
10 g NZ amine
5 g NaCl
2 g MgSO4*7H2O
1. Shake until the solutes have dissolved.
2. Adjust the pH to 7.0 with 5 N NaOh (~0.2 ml).
3. Adjust the volume of the solution to 1 liter with deionized H2O.
4. Sterilize by autoclaving for 20 minutes at 15 lb/sq in on liquid cycle.
50X TAE
For 1 Liter:
242 g tris
57.1 ml concentrated acetic acid
18.61 g EDTA
5X TBE
For 2 Liters:
108 g tris base
55 g boric acid
9.3 g EDTA
pH and filter
pH should be 8.3-8.5
Store in plastic container
10X TBE
For 1 Liter:
108 g tris base
55 g boric acid
9.3 g EDTA
pH and filter
pH should be 8.3-8.5
Store in plastic container
Terrific Broth
For 1 Liter:
900 ml dH2O
12 g bacto-tryptone
24 g bacto-yeast extract
4 ml glycerol
1. Shake until the solutes have dissolved.
2. Sterilize by autoclaving for 20 minutes at 15 lb/sq in on liquid cycle.
3. Allow the solution to cool to 60 degrees or less.
4. Add 100 ml of sterile solutions of 0.17M KH2PO4, 0.72M K2HPO4.
5. After the salts have been dissolved, adjust the volume of the solution to 100 ml with deionized water.
6. Sterilize by autoclaving for 20 minutes at 15 lb/sq in on liquid cycle.
Tetracycline Plates
For 1 Liter:
1 L LB
16 g bacto-agar
1. Autoclave
2. Add 1.25 ml tetra cycline (10 mg/ml in 50% EtOH) to cooled (but not solid) LB/agar
3. Pour plates.
4. Cover with foil and allow to sit overnight and store in cold room.
Note: These plates are light sensitive.
Buffer A (10X)
For 100 ml:
7.0125 g 1.2 M NaCl
0.3728 g 50 mM KCl
0.246 g 10 mM MgSO4.7H2O
0.147 g 10 mM CaCl2.2H2O
1.066 g 50 mM BES (pK = 7.1)
Adjust pH with NaOH to 7.4
Buffer B (10X)
1.802 g 100 mM glucose
0.21 g 25 mM NaHCO3
Adjust pH to 7.4 with HCl
Mix A and B with water to make 1X Ringer solution
Buffer H
50 mM beta-glycerophosphate (FW=216)
1.5 mM EGTA (FW=380)
0.1 mM Na3VO4(FWW=184)
1 mM DTT
The buffer can be stored at -20.
Add protease inhibitors right before use.
Keywords.
Adenylyl cyclase (AC), amyloid precursor protein (APP), Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), cAMP, cAMP-responsive element (CRE), CREB, extra-cellular regulated protein kinase (ERK), FE65, long-term potentiation (LTP), long-term depression (LTD), memory formation, neuroplasticity, local protein synthesis.