BioProtocols--sol5

Gel loading buffer Type I (with sucrose)(6X)(10 ml)

1. Weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF and 4 g sucrose.

2. Make up volume to 10 ml with distilled water.
Store at 4 C.
<USAGE> Bromophenol blue migrates through agarose gels approximately 2.2 times faster than xylene cyanol FF. Bromophenol blue migrates through agarose gels run in 0.5X TBE at approximately the same rate as linear double-stranded DNA 300 bp in length, whereas xylene cyanol FF migrates at approximately the same rate as linear double-stranded DNA 4kb in length. These relationships are most significantly affected by the concentration of agarose in the gel over the range of 0.5% to 1.4%. The choice of loading dye is a matter of personal preference. Bromocresol green should be used as a tracking dye in alkaline gels because it displays a more vivid colour than bromophenol blue at alkaline pH.

Gel loading buffer Type II (with Ficoll)(6X)(10 ml)

1. Weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF and 1.5 g Ficoll (Type 400; Pharmacia).

2. Make up volume to 10 ml with distilled water.
<STORAGE> Store at 4 C.
<USAGE> See Gel loading buffer type I.

Gel loading buffer Type III (withglycerol)(6 X)(10 ml)

1. Weigh out 25 mg bromophenol blue and 25 mg xylene cyanol FF

2. Add 3 ml glycerol.

3. Make up volume to 10 ml with distilled water.
<STORAGE> Store at 4 C.
<USAGE> See Gel loading buffer type I.

Gel loading buffer Type IV (bromophenol blue and sucrose)(6X)(10 ml)

1. Weigh out 25 mg bromophenol blue and 4 g sucrose.

2. Make up volume to 10 ml with distilled water.
<STORAGE> Store at 4 C.
<USAGE> See Gel loading buffer type I.

Gel loading buffer Type V (Alkaline loading buffer)

1. Weigh out 15 mg bromocresol green and 25 mg xylene cyanol FF.

2. Add 120 ul 0.5 M EDTA (pH 8.0) and 3 ml 1 M NaOH.

3. Make up volume to 10 ml with distilled water.
<STORAGE> Store at 4 C.
<USAGE> See Gel loading buffer type I.

10X Phosphate buffered saline (10X PBS)(1 liter)

1. Weigh out 2 g KCl, 80 g NaCl, 17.8 g Na2HPO4.2H2O and 2.4 g KH2PO4.

2. Add 800 ml distilled water to dissolve.

3. Make up volume to 1 liter with distilled water.

4. Dispense into aliquots and sterilize by autoclaving.
<USAGE> Dilute 1:9 with distilled water. Adjust to pH 7.4 with KOH.

Microtubules stabilizing buffer (MTSB)(1 liter)

1. Weigh out 15 g PIPES, 1.9 g EGTA, 1.32 g MgSO4.7H2O and 5 g KOH.

2. Add 800 ml distilled water to dissolve.

3. Adjust to pH 7.0 with KOH.

4. Make up volume to 1 liter with distilled water.

5. Dispense into aliquots and sterilize by autoclaving.
<USAGE> Dilute 1:9 with distilled water. This is an alternative to PBS for use in more sensitive applications.

Ampicillin stock solution (25 mg ml-1)(40 ml)

1. Weigh out 1 g ampicillin (sodium salt) and dissolve in 35 ml of distilled water.

2. Make up volume to 40 ml with distilled water.

3. Sterilize by filtration.
<STORAGE> Aliquots of appropriate volume should be stored at -20 C.
For culture plates, allow media to cool to 55 C before adding ampicillin to a final concentration of 35-50 ug/ml.

Chloramphenical stock solution (34 mg ml-1)(29.5 ml)

1. Weigh out 1 g chloramphenicol and dissolve in 25 ml of 100% ethanol.

2. Make up volume to 29.5 ml with 100% ethanol.
<STORAGE> Aliquots of appropriate volume can be stored at -20 C for a year.
<USAGE> For culture plates, allow media to cool to 55 C before adding chloramphenicol to a final concentration of 10 ug/ml.

Kanamycin stock solution (25 mg ml-1)(40 ml)

1. Weigh out 1 g kanamycin sulfate and dissolve in 35 ml of distilled water.

2. Make up volume to 40 ml with distilled water.

Sterilise by filtration.
<STORAGE> Aliquots of appropriate volume can be stored at -20 C.
<USAGE> For culture plates, allow media to cool to 55 C before adding kanamycin to a final concentration of 50 ug/ml.

Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )