Griffitts:Stock solutions

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(Antibiotics: Added stock concentrations to recipes)
(Gentamicin (40 mL): changed to 20 mL)
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* Do '''not''' filter sterilize—the ethanol may dissolve the filter
* Do '''not''' filter sterilize—the ethanol may dissolve the filter
NOTE: Store at –20°C
NOTE: Store at –20°C
-
===Gentamicin (40 mL)===
+
===Gentamicin (20 mL)===
For a stock solution of 7.5 mg/mL:
For a stock solution of 7.5 mg/mL:
-
* 300 mg [[Wikipedia:Gentamicin|Gentamicin]]
+
* 150 mg [[Wikipedia:Gentamicin|Gentamicin]]
-
* 40 mL ddH<sub>2</sub>O
+
* 20 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
NOTE: Store at 4°C
NOTE: Store at 4°C
 +
===Hygromycin (40 mL)===
===Hygromycin (40 mL)===
For a stock solution of 5 mg/mL:
For a stock solution of 5 mg/mL:
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NOTE: Store at 4°C
NOTE: Store at 4°C
===Nalidixic acid (30 mL)===
===Nalidixic acid (30 mL)===
-
For a stock solution of 5 mg/mL:
+
For a stock solution of 30 mg/mL:
* 900 mg [[Wikipedia:Nalidixic Acid|Nalidixic Acid]]
* 900 mg [[Wikipedia:Nalidixic Acid|Nalidixic Acid]]
* 360 mg NaOH
* 360 mg NaOH
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NOTE: Store at 4°C
NOTE: Store at 4°C
===Streptomycin (40 mL)===
===Streptomycin (40 mL)===
-
For a stock solution of 25 mg/mL:
+
For a stock solution of 50 mg/mL:
* 2 g [[Wikipedia:Streptomycin|Streptomycin]]
* 2 g [[Wikipedia:Streptomycin|Streptomycin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
NOTE: Store at 4°C
NOTE: Store at 4°C
 +
===Tetracycline (40 mL)===
===Tetracycline (40 mL)===
For a stock solution of 2.5 mg/mL:
For a stock solution of 2.5 mg/mL:
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===Triclosan (40 mL)===
===Triclosan (40 mL)===
For a stock solution of 10 mg/mL:
For a stock solution of 10 mg/mL:
-
* 400 mg [[Wikipedia:Tetracycline|Tetracycline]]
+
* 400 mg [[Wikipedia:Triclosan|Triclosan]]
* 40 mL 100% [[Wikipedia:Ethanol|Ethanol]]
* 40 mL 100% [[Wikipedia:Ethanol|Ethanol]]
NOTE: Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter<br>
NOTE: Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter<br>
NOTE: Store at 4°C
NOTE: Store at 4°C
 +
===Trimethoprim (40 mL)===
===Trimethoprim (40 mL)===
For a stock solution of 6.25 mg/mL:
For a stock solution of 6.25 mg/mL:
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! width="120" style="background:#efefef;" | ''S. meliloti''
! width="120" style="background:#efefef;" | ''S. meliloti''
! width="90" style="background:#efefef;" | Solvent
! width="90" style="background:#efefef;" | Solvent
-
! width="60" style="background:#efefef;" | Storage
+
! width="90" style="background:#efefef;" | Storage
|-
|-
| Ampicillin
| Ampicillin
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| Na
| Na
| <center>(none)</center>
| <center>(none)</center>
-
| 5 mg/mL
+
| 30 mg/mL
| 30 μg/mL (1000X)
| 30 μg/mL (1000X)
| N/A
| N/A
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! style="background:#FF0000;" | <font color=white>'''RED'''</font>
! style="background:#FF0000;" | <font color=white>'''RED'''</font>
| 2.5 mg/mL
| 2.5 mg/mL
-
| 10 μg/mL (250X)
+
| 5 μg/mL (500X)
-
| 10 μg/mL (250X)
+
| 5 μg/mL (500X)
| 50% EtOH
| 50% EtOH
| 4°C
| 4°C
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* 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]]
* 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]]
===DNA ladder===
===DNA ladder===
-
* 147 μL ddH<sub>2</sub>0
+
* 140 μL ddH<sub>2</sub>0
-
* 34 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer
+
* 40 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer
* 20 μL 1 kb ladder from NEB
* 20 μL 1 kb ladder from NEB
* 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br>
* 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br>
Note: use 5 μL per lane; 3kb band = 60 ng
Note: use 5 μL per lane; 3kb band = 60 ng
 +
===Ethidium Bromide (1 mL)===
===Ethidium Bromide (1 mL)===
* 100 μL 10 mg/mL ethidium bromide
* 100 μL 10 mg/mL ethidium bromide
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===1 M Sodium Citrate (40 mL)===
===1 M Sodium Citrate (40 mL)===
-
* 40 mL ddH<sub>2</sub>O
+
* 35 mL ddH<sub>2</sub>O
* 11.76  g sodium citrate tribasic dihydrate (Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>·2H<sub>2</sub>O)
* 11.76  g sodium citrate tribasic dihydrate (Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>·2H<sub>2</sub>O)
-
* pH to 5.7 with HCl
+
* pH to 5.7 with HCl (~1 mL)
 +
* QS to 40 mL with ddH<sub>2</sub>O
Sterile filter
Sterile filter

Revision as of 11:36, 19 March 2013

Contents

Antibiotics

Ampicillin (40 mL)

For a stock solution of 25 mg/mL:

NOTE: Store at 4°C

Chloramphenicol (40 mL)

For a stock solution of 7.5 mg/mL:

NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C

Erythromycin (20 mL)

For a stock solution of 10 mg/mL:

  • 200 mg Erythromycin
  • 20 mL 100% ethanol (in fume hood)
  • Do not filter sterilize—the ethanol may dissolve the filter

NOTE: Store at –20°C

Gentamicin (20 mL)

For a stock solution of 7.5 mg/mL:

NOTE: Store at 4°C

Hygromycin (40 mL)

For a stock solution of 5 mg/mL:

NOTE: Store at 4°C

Kanamycin (40 mL)

For a stock solution of 7.5 mg/mL:

  • 300 mg Kanamycin
  • 40 mL ddH2O
  • Filter sterilize

NOTE: Store at 4°C

Nalidixic acid (30 mL)

For a stock solution of 30 mg/mL:

NOTE: Store at 4°C

Neomycin (40 mL)

For a stock solution of 25 mg/mL:

  • 1 g Neomycin
  • 40 mL ddH2O
  • Filter sterilize

NOTE: Store at 4°C

Phosphomycin

For a stock solution of 25 mg/mL:

NOTE: Store at 4°C

Rifampicin (20 mL)

For a stock solution of 50 mg/mL:

  • 1 g Rifampicin
  • 20 mL N,N-dimethylformamide (DMF; in fume hood)

NOTE: Do not filter sterilize—the DMF will dissolve the filter
NOTE: Store at –20°C

Spectinomycin (40 mL)

For a stock solution of 20 mg/mL:

NOTE: Store at 4°C

Streptomycin (40 mL)

For a stock solution of 50 mg/mL:

NOTE: Store at 4°C

Tetracycline (40 mL)

For a stock solution of 2.5 mg/mL:

NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C

Triclosan (40 mL)

For a stock solution of 10 mg/mL:

NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C

Trimethoprim (40 mL)

For a stock solution of 6.25 mg/mL:

NOTE: Do not filter sterilize—the DMSO may dissolve the filter
NOTE: Store at 4°C

Antibiotics Table

Antibiotic Specifications
Antibiotic Abbr. Stripe Stock E. coli S. meliloti Solvent Storage
Ampicillin Ap BLACK 25 mg/mL 100 μg/mL (250X) N/A ddH2O 4°C
Chloramphenicol Cm BROWN 7.5 mg/mL 30 μg/mL (250X) N/A 50% EtOH 4°C
Erythromycin Er
(none)
10 mg/mL N/A N/A 100% EtOH –20°C
Gentamycin Gm PURPLE 7.5 mg/mL 3 μg/mL (2500X) 15 μg/mL (500X) ddH2O 4°C
Hygromycin Hy
(none)
5 mg/mL 20 μg/mL (250X) 20 μg/mL (250X) ddH2O 4°C
Kanamycin Km YELLOW 7.5 mg/mL 30 μg/mL (250X) Use Neomycin ddH2O 4°C
Nalidixic acid Na
(none)
30 mg/mL 30 μg/mL (1000X) N/A ddH2O 4°C
Neomycin Nm GREEN 25 mg/mL Use Kanamycin 100 μg/mL (250X) ddH2O 4°C
Phosphomycin Pm
(none)
25 mg/mL N/A 100 μg/mL (250X) ddH2O 4°C
Rifampicin Rf
(none)
50 mg/mL N/A 100 μg/mL (500X) DMF –20°C
Spectinomycin Sp ORANGE 20 mg/mL 80 μg/mL (250X) 80 μg/mL (250X) ddH2O 4°C
Streptomycin Sm BLUE 50 mg/mL N/A 200 μg/mL (250X) ddH2O 4°C
Tetracycline Tc RED 2.5 mg/mL 5 μg/mL (500X) 5 μg/mL (500X) 50% EtOH 4°C
Triclosan Tr
(none)
10 mg/mL N/A N/A 100% EtOH 4°C
Trimethoprim Tm
(none)
6.25 mg/mL N/A 12.5 μg/mL (500X)
or
25 μg/mL (250X)
100% DMSO 4°C

Note: These parameters are intended to be compatible with pre-made 250-mL amounts of LB agar, such that usually 1 mL of stock solution is diluted into the LB agar. For liquid LB (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin.

DNA

10 mM dNTPs

  • Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
  • Master Mix:
  • Dispense 100-μL aliquots into 1.7-mL tubes

Note: Store in the –20°C freezer in the box marked "Other Enzymes"

10X Taq buffer

  • Label tubes: 10X Taq buffer
  • Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
  • Aliquot 375 μL of "Thermo pol buffer" into tubes—do not dilute!

Note: Store in the –20°C freezer in the box marked "Other Enzymes"

10X Pfx buffer

  • Label tubes: 10X Pfx buffer
  • Thaw and mix (stir) "Pfx50 10X PCR Buffer" (NEB; in freezer)
  • Aliquot 375 μL of "Pfx50 10X PCR Buffer" into tubes—do not dilute!

Note: Store in the –20°C freezer in the box marked "Other Enzymes"

8X DNA loading

DNA ladder

Note: use 5 μL per lane; 3kb band = 60 ng

Ethidium Bromide (1 mL)

  • 100 μL 10 mg/mL ethidium bromide
    • This is stored on the middle shelf of the refrigerator on the right
  • 900 μL ddH2O

Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide

Ethidium Bromide (10 mL)

  • 10 mL ddH2O
  • 10 mg ethidium bromide

Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide

Media Additives

1 M Calcium Chloride (75 mL)

  • 75 mL ddH2O
  • 11.027 g calcium chloride dihydrate (CaCl2·2H2O)

Autoclave

2 M Magnesium Chloride (75 mL)

  • 75 mL ddH2O
  • 30.4965 g magnesium chloride hexahydrate (MgCl2·6H2O)

Autoclave

1 M Sodium Citrate (40 mL)

  • 35 mL ddH2O
  • 11.76 g sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O)
  • pH to 5.7 with HCl (~1 mL)
  • QS to 40 mL with ddH2O

Sterile filter

2% X-gal

  • 10 mL N,N-dimethylformamide (DMF)(in fume hood)
  • 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer)

Store at –20°C

0.2 M IPTG

  • 5 mL ddH2O
  • 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)

Sterile filter, aliquot, and store at –20°C

1 M Copper (II) sulfate (50 mL)

  • 50 mL ddH2O
  • 7.98 g CuSO4

Sterile filter

10% SDS (75 mL)

  • 75 mL ddH2O
  • 7.5 g Sodium dodecyl sulfate (SDS)

Do not sterilize

20% SDS (75 mL)

  • 75 mL ddH2O
  • 15 g Sodium dodecyl sulfate (SDS)

Do not sterilize

30% Acrylamide (75 mL)

  • 75 mL ddH2O
  • 22.5 g acrylamide

40% Acrylamide (75 mL)

  • 75 mL ddH2O
  • 30 g acrylamide

10% Ammonium persulfate (75 mL)

  • 75 mL ddH2O
  • 7.5 g ammonium persulfate (APS)

5 M NaCl (75 mL)

  • 75 mL ddH2O
  • 21.92 g NaCl

Autoclave

80% Glycerol (75 mL)

  • 15 mL ddH2O
  • 60 mL glycerol

Microwave for 30-second intervals on high until dissolved
Autoclave

1 M DTT (5 mL)

  • 5 mL ddH2O
  • 771 mg Dithiothreitol (DTT; in fridge)

Aliquot 500 μL into Eppendorf tubes Store in –20°C

Orcinol spray

  • 5 mL ddH2O
  • 25 mL methanol (in fume hood)
  • 10 mL sulfuric acid (H2SO4 (in fume hood—be careful!)
  • 200 mg orcinol
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