Griffitts:Stock solutions

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Antibiotics: added a table)
m (Triclosan (40 mL))
(46 intermediate revisions not shown.)
Line 1: Line 1:
-
{{Griffitts}}
+
<div class="noprint">
 +
{{Template:Griffitts}}
 +
</div>
==Antibiotics==
==Antibiotics==
===Ampicillin (40 mL)===
===Ampicillin (40 mL)===
 +
For a stock solution of 25 mg/mL:
* 1 g [[Wikipedia:Ampicillin|Ampicillin]]
* 1 g [[Wikipedia:Ampicillin|Ampicillin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
===Chloramphenicol (40 mL)===
===Chloramphenicol (40 mL)===
-
* 0.3 g [[Wikipedia:Chloramphenicol|Chloramphenicol]]
+
For a stock solution of 7.5 mg/mL:
-
* 40 mL [[Wikipedia:Ethanol|Ethanol]]
+
* 300 mg [[Wikipedia:Chloramphenicol|Chloramphenicol]]
-
** Do '''not''' filter sterilize
+
* 40 mL 50% [[Wikipedia:Ethanol|Ethanol]]
-
** Store in refrigerator
+
NOTE: Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter<br>
-
===Gentamycin (40 mL)===
+
NOTE: Store at 4°C
-
* 0.3 g [[Wikipedia:Gentamycin|Gentamycin]]
+
===Erythromycin (20 mL)===
 +
For a stock solution of 10 mg/mL:
 +
* 200 mg [[Wikipedia:Erythromycin|Erythromycin]]
 +
* 20 mL 100% ethanol (in fume hood)
 +
* Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter
 +
NOTE: Store at &ndash;20°C
 +
===Gentamicin (40 mL)===
 +
For a stock solution of 7.5 mg/mL:
 +
* 300 mg [[Wikipedia:Gentamicin|Gentamicin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
===Hygromycin (40 mL)===
===Hygromycin (40 mL)===
 +
For a stock solution of 5 mg/mL:
 +
* 200 mg [[Wikipedia:Hygromycin|Hygromycin]]
 +
* 40 mL ddH<sub>2</sub>O
 +
* Filter sterilize
 +
NOTE: Store at 4°C
===Kanamycin (40 mL)===
===Kanamycin (40 mL)===
-
* 0.1 g [[Wikipedia:Kanamycin|Kanamycin]]
+
For a stock solution of 7.5 mg/mL:
 +
* 300 mg [[Wikipedia:Kanamycin|Kanamycin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
 +
===Nalidixic acid (30 mL)===
 +
For a stock solution of 30 mg/mL:
 +
* 900 mg [[Wikipedia:Nalidixic Acid|Nalidixic Acid]]
 +
* 360 mg NaOH
 +
* 30 mL ddH<sub>2</sub>O
 +
* Filter sterilize
 +
NOTE: Store at 4°C
===Neomycin (40 mL)===
===Neomycin (40 mL)===
-
* 0.8 g [[Wikipedia:Neomycin|Neomycin]]
+
For a stock solution of 25 mg/mL:
 +
* 1 g [[Wikipedia:Neomycin|Neomycin]]
 +
* 40 mL ddH<sub>2</sub>O
 +
* Filter sterilize
 +
NOTE: Store at 4°C
 +
===Phosphomycin===
 +
For a stock solution of 25 mg/mL:
 +
* 1 g [[Wikipedia:Fosfomycin|Phosphomycin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
 +
===Rifampicin (20 mL)===
 +
For a stock solution of 50 mg/mL:
 +
* 1 g [[Wikipedia:Rifampicin|Rifampicin]]
 +
* 20 mL N,N-dimethylformamide (DMF; in fume hood)
 +
NOTE: Do '''not''' filter sterilize&mdash;the DMF will dissolve the filter<br>
 +
NOTE: Store at &ndash;20°C
===Spectinomycin (40 mL)===
===Spectinomycin (40 mL)===
-
* 20 mg [[Wikipedia:Spectinomycin|Spectinomycin]]
+
For a stock solution of 20 mg/mL:
 +
* 800 mg [[Wikipedia:Spectinomycin|Spectinomycin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
===Streptomycin (40 mL)===
===Streptomycin (40 mL)===
-
* 2.0 g [[Wikipedia:Streptomycin|Streptomycin]]
+
For a stock solution of 50 mg/mL:
 +
* 2 g [[Wikipedia:Streptomycin|Streptomycin]]
* 40 mL ddH<sub>2</sub>O
* 40 mL ddH<sub>2</sub>O
* Filter sterilize
* Filter sterilize
-
** Store in refrigerator
+
NOTE: Store at 4°C
 +
 
===Tetracycline (40 mL)===
===Tetracycline (40 mL)===
-
* 0.1 g [[Wikipedia:Tetracycline|Tetracycline]]
+
For a stock solution of 2.5 mg/mL:
-
* 40 mL [[Wikipedia:Ethanol|Ethanol]]
+
* 100 mg [[Wikipedia:Tetracycline|Tetracycline]]
-
** Do '''not''' filter sterilize
+
* 40 mL 50% [[Wikipedia:Ethanol|Ethanol]]
-
** Store in refrigerator
+
NOTE: Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter<br>
-
<br>
+
NOTE: Store at 4°C
-
<br>
+
===Triclosan (40 mL)===
-
<br>
+
For a stock solution of 10 mg/mL:
 +
* 400 mg [[Wikipedia:Triclosan|Triclosan]]
 +
* 40 mL 100% [[Wikipedia:Ethanol|Ethanol]]
 +
NOTE: Do '''not''' filter sterilize&mdash;the ethanol may dissolve the filter<br>
 +
NOTE: Store at 4°C
 +
 
 +
===Trimethoprim (40 mL)===
 +
For a stock solution of 6.25 mg/mL:
 +
* 250 mg [[Wikipedia:Trimethoprim|Trimethoprim]]
 +
* 40 mL 100% [[Wikipedia:Dimethyl sulfoxide|DMSO]]
 +
NOTE: Do '''not''' filter sterilize&mdash;the DMSO may dissolve the filter<br>
 +
NOTE: Store at 4°C
 +
 
 +
==Antibiotics Table==
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable"
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable"
|+'''Antibiotic Specifications'''
|+'''Antibiotic Specifications'''
Line 54: Line 107:
! width="120" style="background:#efefef;" | ''E. coli''
! width="120" style="background:#efefef;" | ''E. coli''
! 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;" | Storage
|-
|-
| Ampicillin
| Ampicillin
Line 61: Line 116:
| 100 μg/mL (250X)
| 100 μg/mL (250X)
| N/A
| N/A
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Chloramphenicol
| Chloramphenicol
Line 68: Line 125:
| 30 μg/mL (250X)
| 30 μg/mL (250X)
| N/A
| N/A
 +
| 50% EtOH
 +
| 4°C
 +
|-
 +
| Erythromycin
 +
| Er
 +
| <center>(none)</center>
 +
| 10 mg/mL
 +
| N/A
 +
| N/A
 +
| 100% EtOH
 +
| &ndash;20°C
|-
|-
| Gentamycin
| Gentamycin
Line 73: Line 141:
! style="background:#9400D3;" | <font color=white>'''PURPLE'''</font>
! style="background:#9400D3;" | <font color=white>'''PURPLE'''</font>
| 7.5 mg/mL
| 7.5 mg/mL
-
| 3 μg/mL (250X)
+
| 3 μg/mL (2500X)
| 15 μg/mL (500X)
| 15 μg/mL (500X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Hygromycin
| Hygromycin
| Hy
| Hy
-
| (none)
+
| <center>(none)</center>
| 5 mg/mL
| 5 mg/mL
| 20 μg/mL (250X)
| 20 μg/mL (250X)
| 20 μg/mL (250X)
| 20 μg/mL (250X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Kanamycin
| Kanamycin
Line 88: Line 160:
| 7.5 mg/mL
| 7.5 mg/mL
| 30 μg/mL (250X)
| 30 μg/mL (250X)
 +
| '''Use Neomycin'''
 +
| ddH<sub>2</sub>O
 +
| 4°C
 +
|-
 +
| Nalidixic acid
 +
| Na
 +
| <center>(none)</center>
 +
| 30 mg/mL
 +
| 30 μg/mL (1000X)
| N/A
| N/A
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Neomycin
| Neomycin
| Nm
| Nm
! style="background:#008000;" | <font color=white>'''GREEN'''</font>
! style="background:#008000;" | <font color=white>'''GREEN'''</font>
-
| 20 mg/mL
+
| 25 mg/mL
 +
| '''Use Kanamycin'''
 +
| 100 μg/mL (250X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
 +
|-
 +
| Phosphomycin
 +
| Pm
 +
| <center>(none)</center>
 +
| 25 mg/mL
| N/A
| N/A
-
| 80 μg/mL (250X)
+
| 100 μg/mL (250X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
 +
|-
 +
| Rifampicin
 +
| Rf
 +
| <center>(none)</center>
 +
| 50 mg/mL
 +
| N/A
 +
| 100 μg/mL (500X)
 +
| DMF
 +
| &ndash;20°C
|-
|-
| Spectinomycin
| Spectinomycin
Line 103: Line 206:
| 80 μg/mL (250X)
| 80 μg/mL (250X)
| 80 μg/mL (250X)
| 80 μg/mL (250X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Streptomycin
| Streptomycin
Line 110: Line 215:
| N/A
| N/A
| 200 μg/mL (250X)
| 200 μg/mL (250X)
 +
| ddH<sub>2</sub>O
 +
| 4°C
|-
|-
| Tetracycline
| Tetracycline
Line 115: Line 222:
! 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
 +
| 4°C
 +
|-
 +
| Triclosan
 +
| Tr
 +
| <center>(none)</center>
 +
| 10 mg/mL
 +
| N/A
 +
| N/A
 +
| 100% EtOH
 +
| 4°C
 +
|-
 +
| Trimethoprim
 +
| Tm
 +
| <center>(none)</center>
 +
| 6.25 mg/mL
 +
| N/A
 +
| 12.5 μg/mL (500X)<br><center>or</center>25 μg/mL (250X)
 +
| 100% DMSO
 +
| 4°C
|}
|}
-
Note: These parameters are intended to be compatible with pre-made 250-mL amounts of [[Griffitts:Common media#LB Broth (1 L)|LB]] agar, such that usually 1 mL of stock solution is diluted into the [[Griffitts:Common media#LB Broth (1 L)|LB]] agar. For liquid [[Griffitts:Common media#LB Broth (1 L)|LB]] (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exception: Gentamycin for ''E. coli''.
+
Note: These parameters are intended to be compatible with pre-made 250-mL amounts of [[Griffitts:Common media#LB Broth (1 L)|LB]] agar, such that usually 1 mL of stock solution is diluted into the [[Griffitts:Common media#LB Broth (1 L)|LB]] agar. For liquid [[Griffitts:Common media#LB Broth (1 L)|LB]] (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin.
==DNA==
==DNA==
===10 mM dNTPs===
===10 mM dNTPs===
-
* Thaw and mix (stir) 100 mM stock solutions of G, A, T, C (NEB)
+
* Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
-
* Combine in a 1.5-mL tube:
+
* Master Mix:
-
** 60 μl H<sub>2</sub>O
+
** 60 μl sterile dH<sub>2</sub>O
-
** 10 μl [[Wikipedia:Guanine|G]]
+
** 10 μl [[Wikipedia:Guanine|dGTP]]
-
** 10 μl [[Wikipedia:Adenine|A]]
+
** 10 μl [[Wikipedia:Adenine|dATP]]
-
** 10 μl [[Wikipedia:Thymine|T]]
+
** 10 μl [[Wikipedia:Thymine|dTTP]]
-
** 10 μl [[Wikipedia:Cytosine|C]]
+
** 10 μl [[Wikipedia:Cytosine|dCTP]]
 +
* Dispense 100-μL aliquots into 1.7-mL tubes
 +
Note: Store in the &ndash;20°C freezer in the box marked "Other Enzymes"
===10X ''Taq'' buffer===
===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&mdash;do '''not''' dilute!
 +
Note: Store in the &ndash;20°C freezer in the box marked "Other Enzymes"
===10X ''Pfx'' buffer===
===10X ''Pfx'' buffer===
-
===20X Triton X-100===
+
* 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&mdash;do '''not''' dilute!
 +
Note: Store in the &ndash;20°C freezer in the box marked "Other Enzymes"
 +
===8X DNA loading===
 +
* 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]]
 +
* 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]]
 +
===DNA ladder===
 +
* 140 μL ddH<sub>2</sub>0
 +
* 40 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer
 +
* 20 μL 1 kb ladder from NEB
 +
* 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
 +
 
 +
===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 ddH<sub>2</sub>O
 +
Note: use 8 μL per 50 mL gel<br>
 +
WARNING: Always wear gloves when handling ethidium bromide
 +
 
 +
===Ethidium Bromide (10 mL)===
 +
* 10 mL ddH<sub>2</sub>O
 +
* 10 mg ethidium bromide<br>
 +
Note: use 8 μL per 50 mL gel<br>
 +
WARNING: Always wear gloves when handling ethidium bromide
 +
 
==Media Additives==
==Media Additives==
===1 M Calcium Chloride (75 mL)===
===1 M Calcium Chloride (75 mL)===
-
* 75 mL dH<sub>2</sub>O
+
* 75 mL ddH<sub>2</sub>O
-
* 11.027 g CaCl<sub>2</sub>·2H<sub>2</sub>O
+
* 11.027 g calcium chloride dihydrate (CaCl<sub>2</sub>·2H<sub>2</sub>O)
Autoclave
Autoclave
-
===X-gal===
+
===2 M Magnesium Chloride (75 mL)===
-
===IPTG===
+
* 75 mL ddH<sub>2</sub>O
-
===Copper sulfate===
+
* 30.4965 g magnesium chloride hexahydrate (MgCl<sub>2</sub>·6H<sub>2</sub>O)
-
===SDS===
+
Autoclave
 +
 
 +
===1 M Sodium Citrate (40 mL)===
 +
* 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)
 +
* pH to 5.7 with HCl (~1 mL)
 +
* QS to 40 mL with ddH<sub>2</sub>O
 +
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 &ndash;20°C
 +
 
 +
===0.2 M IPTG===
 +
* 5 mL ddH<sub>2</sub>O
 +
* 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)
 +
Sterile filter, aliquot, and store at &ndash;20°C
 +
 
 +
===1 M Copper (II) sulfate (50 mL)===
 +
* 50 mL ddH<sub>2</sub>O
 +
* 7.98 g CuSO<sub>4</sub>
 +
Sterile filter
 +
 
 +
===10% [[SDS]] (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 7.5 g Sodium dodecyl sulfate ([[SDS]])
 +
Do '''not''' sterilize
 +
===20% [[SDS]] (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 15 g Sodium dodecyl sulfate ([[SDS]])
 +
Do '''not''' sterilize
 +
 
 +
===30% Acrylamide (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 22.5 g acrylamide
 +
 
 +
===40% Acrylamide (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 30 g acrylamide
 +
 
 +
===10% Ammonium persulfate (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 7.5 g ammonium persulfate (APS)
 +
 
 +
===5 M NaCl (75 mL)===
 +
* 75 mL ddH<sub>2</sub>O
 +
* 21.92 g NaCl
 +
Autoclave
 +
 
 +
===80% Glycerol (75 mL)===
 +
* 15 mL ddH<sub>2</sub>O
 +
* 60 mL glycerol
 +
Microwave for 30-second intervals on high until dissolved<br>
 +
Autoclave
 +
 
 +
===1 M DTT (5 mL) ===
 +
* 5 mL ddH<sub>2</sub>O
 +
* 771 mg Dithiothreitol (DTT; in fridge)
 +
Aliquot 500 μL into Eppendorf tubes
 +
Store in &ndash;20°C
 +
 
 +
===Orcinol spray===
 +
* 5 mL ddH<sub>2</sub>O
 +
* 25 mL methanol (in fume hood)
 +
* 10 mL sulfuric acid (H<sub>2</sub>SO<sub>4</sub> (in fume hood&mdash;be careful!)
 +
* 200 mg orcinol

Revision as of 18:41, 29 February 2012

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 (40 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
Personal tools