|
Jackson's Laboratory Other Links |
Commonly Used Reagents |
||
|
To view the recipe for a specific reagent, scroll down to find the reagent or click on the link below that corresponds to the first letter of the reagent |
|||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
|
|
Acrylamide gel for sequencing |
29.42
grams Urea 10.5mL
Acryl bis (19:1) 10mL
TBE (1X) 25mL
H2O Mix all ingredients and filter through a Millipore filter unit. Add 330m L of 10% ammonium persulfate (APS) [e.g., 60m g APS in 600m L H2O]. Add 33m L TMED. Swirl to mix and pour immediately. |
|
|
Agarose gel |
Add the desired amount of agarose
(Molecular Biology Grade) to a volume of running buffer (1X TAE or 1X TBE) sufficient
for constructing a gel. For example, for 1% agarose add 1 gram of agarose per
100mL of buffer. Melt the agarose in a microwave
(swirl occasionally to ensure even mixing). Gel solution will be EXTREMELY
HOT! Replace the lost volume (due to boiling)
with deionized water (NOTE: mixture must be preweighed). Add 3m L 10mg/mL ethidium bromide to each 100ml of gel solution. |
|
|
Alkaline Lysis Buffers, plasmid
preps |
Solution I 50mM glucose 25mM Tris-HCl, pH 8.0 10mM EDTA, pH 8.0 This solution can be prepared in
batches of approximately 100ml, autoclaved for 15 minutes, and stored at 4°
C. Solution II 0.2N NaOH (freshly diluted from 10N
stock) 1% SDS Prepare
fresh prior to use. Solution III 60ml 5M Potassium acetate 11.5ml Glacial acetic acid 28.5ml dH2O This solution is 3M with respect
to potassium and 5M with respect to acetate. Ref: Molecular Biology: A Laboratory Manual (1989) Eds: Sambrook et.al. |
|
|
Alsever's Solution |
Dextrose
(20.5 grams) Sodium
citrate (8 grams) Citric
acid (0.55 grams) NaCl
(4.2 grams) Dissolve ingredients successively
in deionized water and autoclave at 15psi for 15 minutes. The pH of the
sterilized solution should be 6.1. Add one part Alsever's solution to one
part whole blood. Allow suspension to stabilize at 4C for a week prior to
use. Alsever's solution is an
isotonic, anticoagulant blood preservative that permits storage of whole
blood at 4C for about 10 weeks. Ref: Methods in Immunology, A Laboratory Test for Instruction and Research. DH Cambell, et al., WA Benjamin, Inc., 1963. Page 244. |
|
|
BSA, 3% |
Add 3 grams of bovine serum
albumin (Fraction V) to 100mL of PBS. Allow to dissolve and store at 4C. Ref: Antibodies, A Laboratory Manual (1988) Harlow and Lane. Cold Spring Harbor Publications. Page 684. |
|
|
CaCl2 (for Ca-PO4 transfection) |
Calcium chloride dihydrate (CaCl2.2H2O,
FW = 147.02) Prepare a 2M solution (10mL). Measure 2.94 grams calcium chloride and transfer to a 15mL conical tube. Add approximately 8mL deionized water. Vortex to dissolve and bring the final volume to 10mL. Filter-sterilize the solution using a syringe filter (0.22m m). Store at 4C. Keep sterile. |
|
|
Citrate Buffer |
16.67mM Na2HPO4
(0.67 grams Na2HPO4.7H2O) 8.33mM Citric Acid (0.26 grams C6H8O7.H2O) Add ingredients to approximately 100mL deionized water. Adjust the pH to 5.0 and bring the final volume to 150mL. |
|
|
|
|
|
|
Ethidium Bromide, 10mg/ml |
Add 10 grams ethidium bromide to 100mL deionized water. Stir on a magnetic stirrer for several hours to ensure that the dye is dissolved. Store in a dark bottle at room temperature. Ethidium bromide is a DNA intercalating dye and a powerful mutagen. Wear gloves when working with solutions containing this dye. |
|
|
|
|
|
|
GBS (Glycine buffered saline) |
0.2M
Glycine (1.5 grams) 0.1M
NaCl (0.58 grams) Add all ingredients to approximately 75mL deionized water. Adjust the pH to 2.5 using HCl. Adjust the final volume to 100mL. |
|
|
Glucose, 1M |
Dissolve 18 grams of glucose in 90mL deionized water. Bring the final volume to 100mL. Filter sterilize using a 0.22m m filter. DO NOT AUTOCLAVE. |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
KCl, 250mM |
Add 1.86 grams KCl to 100mL of deionized water |
|
|
Laemmli Buffer, 5X |
200mM
Tris-Cl, pH 6.8 (5mL of 0.5M stock) 400mM
b -mercaptoethanol (0.375mL of 14% stock) 8%
SDS (0.8 gram) 40%
Glycerol (5mL of 100% stock) 0.4%
Bromophenol blue (1 milligram) Add Tris, b -mercaptoethanol, and glycerol; Mix well. Add SDS. Mix and heat briefly (15-20 seconds) in a microwave to dissolve the SDS. Add the bromophemol blue last. Store at -20C in 1mL aliquots. |
|
|
Luria-Bertani Medium |
Per liter: To 990mL deionized water add 10 grams Bactotryptone 5 grams Bactoyeast Extract 10 grams NaCl Adjust the pH to 7.0 with 5N NaOH. Sterilize by autoclaving. For LB plates, add 15 grams agar per liter. Ref: Molecular Cloning, A Laboratory Manual. Eds. Sambrook, et al. (1989) |
|
|
MgCl2, 2M |
Add 19 grams of MgCl2 to 90mL deioized water. Adjust the solution to 100mL. Sterilize by autoclaving. |
|
|
|
|
|
|
|
|
|
|
PBS (Phosphate buffered saline) |
100mM
NaCl (2.92 grams) 20mM
Na2HPO4 (1.42 grams) 5mM
KH2PO4 (0.34 grams) Add all ingredients to approximately 400mL deionized water. Bring the total volume to 500mL. Sterilize by autoclaving. |
|
|
PBS, 1X |
NaCl
(8 grams) KCl
(0.2 grams) Na2HPO4.12H2O
(2.9 grams) KH2PO4
(0.2 grams) Add all ingredients to approximately 800mL deionized water. Adjust the pH to 7.4. Bring the final volume to 1 liter. Autoclave to sterilize and store at 4C. |
|
|
PBS, 10X |
NaCl
(80 grams) KCl
(2 grams) Na2HPO4,anhydrous
(11.5 grams) KH2PO4
(2 grams) Add all ingredients to approximately
800mL deionized water. Adjust the pH to 7.2. Bring the final volume to 1
liter. Add 0.2 grams Thiomersal as a preservative, if desired (cannot be used
for tissue culture). Store at 4C. This solution is 0.01M with respect to
phosphate and 0.15M with respect to NaCl. Dilute 1:10 prior to use. Ref: Liddell, JE and A. Cryer. A Practical Guide to Monoclonal Antibodies. John Wiley and Sons. Page 156 and 160 (1991). |
|
|
PCR Buffer |
Stock Solutions: 30mM
MgCl2 0.2M
MOPS, pH 7.75 0.5M
KCl Working Solutions: Add 100m
l of each stock solution to a microcentrifuge tube. Bring the final volume to
1000m l. Working
Concentrations: 3mM
MgCl2 20mM
MOPS 50mM
KCl 200m M each dNTP |
|
|
Phosphate Buffer |
Stock solutions Solution A: 0.2M solution of monobasic
sodium phosphate (27.8g in 1000ml) (acid) Solution B: 0.2M solution of
dibasic sodium phosphate (53.65g of Na2HPO4.7H2O
or 71.7g of Na2HPO4.12H2O in 1000ml) (base) To prepare a phosphate buffer at
a specific pH, use the chart to find the volumes of solution A and B needed,
then x ml of A + y ml of B, to make the buffer. x y pH x y pH 93.5 6.5 5.7 45.0 55.0 6.9 92.0 8.0 5.8 39.0 61.0 7.0 90.0 10.0 5.9 33.0 67.0 7.1 87.7 12.3 6.0 28.0 72.0 7.2 85.0 15.0 6.1 23.0 77.0 7.3 81.5 18.5 6.2 19.0 81.0 7.4 77.5 22.5 6.3 16.0 84.0 7.5 73.5 26.5 6.4 13.0 87.0 7.6 68.5 31.5 6.5 10.5 90.5 7.7 62.5 37.5 6.6 8.5 91.5 7.8 56.5 43.5 6.7 7.0 93.0 7.9 51.0 49.0 6.8 5.3 94.7 8.0 To finely adjust the pH, dilute the required solution to the desired pH with the appropriate stock solution. The stock solution to use depends on the pH desired. Stock solution A will lower the pH, while stock solution B will raise the pH. |
|
|
Polybrene, 10mg/mL |
Prepare a stock solution
containing 10 milligrams of polybrene per milliliter of PBS. Filter-sterilize
using a syringe filter (0.22m m). This is a 1000X solution. Polybrene Is a polycation that
aids in binding of retroviral particles to the cell membrane. Ref: Practical Molecular Virology. Methods in Molecular Biology, Volume 8. Ed. MKL Collins. Page 31 (1991). |
|
|
|
|
|
|
RBC Lysis Buffer, pH 7.2 Also Tris buffered ammonium chloride |
Stock Solutions: NH4Cl
(0.83% weight by volume) Tris
(2.06% weight by volume), pH 7.65 Working Solution: 9
volumes NH4Cl 1
volume Tris-Cl, pH 7.65 Filter
sterilize. Ref: Lymphocytes, A Practical Approach. Ed. GGB Klaus, IRL Press. SV Hunt. Isolation of Lymphocytes and Accessory Cells, Page 32. |
|
|
Sequencing gel, see acrylamide gel |
|
|
|
SOC |
Per liter To
900mL deionized water, add 20
grams bactotryptone 5
grams bactoyeast 0.5
grams NaCl Mix and add 10mL of 250mM KCl. Adjust the pH to 7.0 with 5N NaOH. Bring
the final volume to 1 liter and sterilize by autoclaving. Before use add 5mL
of 2M MgCl2 and 20mL of 1M glucose. Store SOC in 1mL aliquots at 4C. |
|
|
Solution I, II, and III, see Alkaline Lysis buffers |
|
|
|
TAE (Busch buffer) |
40mM
Tris 20mM
Sodium acetate 2mM
EDTA Add ingredients to approximately
800mL deionized water. Adjust the pH to 7.5 with glacial acetic acid. Bring
the final volume to 1 liter. For 1 liter of 10X buffer: Tris
(48.25 grams) Sodium
acetate (27.2 grams) EDTA (7.45 grams) |
|
|
TBE, 10X |
121.1
grams Tris 7.4
grams EDTA 55
gram Boric Acid Dissolve the ingredients in approximately 800mL deionized water; pH the solution to 8.3 with dry boric acid. Filter. Makes 1 liter. |
|
|
TBS (Tris buffered saline), 1X |
20mM
Tris (2.42 grams) 0.5M
NaCl (29.22 grams) Dissolve the ingredients in approximately 800mL deionized water. Adjust the pH to 7.5 with concentrated HCl (approximately 1.5mL). Adjust the final volume to 1 liter. |
|
|
TBS, 10X |
12.1
grams Tris 40
grams NaCl Dissolve the ingredients in
approximately 400mL deionized water. Adjust the pH to 7.6 with approximately
19mL concentrated HCl. For 4 liters, add 96.8 grams Tris and 320 grams NaCl |
|
|
TE |
10mM
Tris-Cl, pH 7.4 1mM EDTA, pH 8.0 |
|
|
TE, Low |
3mM
Tris-Cl, pH 7.4 0.2mM
EDTA, pH 8.0 Good storage buffer for DNA. EDTA concentration will not inhibit enzymatic reactions. Will inhibit nuclease activity. Will not interfere with subsequent use of DNA (e.g., restriction digests, ligation, transfection, etc.). |
|
|
Tris |
Tris (hydroxymethyl)-aminomethane |
|
|
TTBS (Tween plus TBS) |
TBS containing 0.2% TWEEN 20
(0.2mL per 100mL TBS) TWEEN - Polyoxyethylene sorbitan monolaurate |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
X-gal reagent (for eukaryotic staining) |
Per milliliter of reagent: 942m L PBS 20m L 200mM K4Fe(CN)6.3H2O 20m
L 200mM K3Fe(CN)6 2m
L 1M MgCl2 Mix by vortexing and add 16m L of 2% X-gal in dimethyl formamide |
|
|
|
|
|
|
|
|
