Materials and recipes

Equipment and tools

 

Micropipettors (In the class cabinet)

GIBCO BRL Horizon 58 minigel apparatus (In the class cabinet)

Power supply unit (In the class cabinet)

Measuring cylinders (In the class cabinet)

2L beaker (Take a beaker from the clean glassware cabinet in Room 131)

Solutions and consumables

Keep the following on an ice bucket (in microfuge tubes)

10X loading buffer  (aliquots are stored in the class box in -20^oC freezer, return unused portions by the end of the lab)

Molecular Weight marker (aliquots of cut lambda Hind III marker is available in the class box in -20^oC freezer)

DNA samples for loading (These are the DNA samples digested by the students.  Keep them in class box and ask them to identify their samples and take them)

To make an Agarose gel:

Dissolve 2 grams of agarose  in 200 mls of 1x TBE buffer by heating in the microwave.  Cool to 40-50°C, pour into the properly assembled gel mold . 

(10X TBE is available in the cold room make an appropriate dilution to make 1X TBE.   Need 50 mls of agarose per one gel unit.  Calculate the amount needed and dissolve 1g of agarose in 100 ml of 1XTBE in a screwcap bottle during the class, using the microwave in room 131.  Cool it to 50°C and ask the students to pour their own gel.)

Need 50 mls of agarose solution per gel.

TBE buffer for electrophoresis

 

Dilute 200 ml of 10X TBE into 2000 ml with milliQ water.

 

10X stock solution is prepared as follows:

10X stock solution, 1 liter:

108 g Tris base (890 mM)

55 g boric acid (890 mM)

40 ml 0.5 M EDTA, pH 8.0

Need 300 ml of buffer per gel rig.  (10X TBE is stored in the cold room.  Make the appropriate dilution for the class using milliQ water)

EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g Na2EDTA×2H2O in 700 ml H2O

Adjust pH to 8.0 with 10 M NaOH (~50 ml)

Add H2O to 1 liter.

(In the cold room, in a Milk bottle)

Sterile microfuge tubes (in the class cabinet)

10X loading buffer

 

20% Ficoll 400

0.1 M Na2EDTA, pH 8

1.0% sodium dodecyl sulfate

0.25% Bromphenol blue

0.25% Xylene Cyanol

Add 1/10 th of the volume of the reaction mix before loading on to the gel.

(aliquots are stored in the class box in -20^oC freezer)

Ethidium bromide, 10 mg/ml

Dissolve 0.2 g ethidium bromide in 20 ml H2O

Mix well and store at 4°C in dark

Final concentration of ethidium bromide should be 0.5 µg/ml.  Add 10 µl of stock solution to the running buffer once the dye front reached half way.  Be careful to unplug the apparatus before adding ethidium bromide.  Mix by gentle swirling and allow it to run for another 45 minutes. 

(aliquot is stored in a labeled glass beaker in the refrigerator on the brown tray, top shelf, on the left.  Be very careful.  This is mutagenic and carcinogenic)

Miscellaneous items