Day 1: Find it, Do it, Teach It!

 

* Perform each of  the 9 basic lab tasks from start to finish

* Focus on the steps to complete tasks

* Document  the steps for developing teaching protocols and assessment, including a rubric as appropriate. The assessment should include both group and individual accountability, and a specific set of questions or measurements to assess performance (How do you know if they're doing it right? How do you know if they're learning what you want them to learn?)

*  Prepare one complete protocol and assessment presentation with images and Power Point instructions.

 

Exercises:

1. pH: using standardized meter, identify pH of unknown; make 1M citrate solution, adjust to 6.5; using Internet - find what buffer is used for; how to standardize, clean probe, how to drop (adjust) the acid/base

2. Molarity:  make 100ml of 0.5 M NaCl solution

3. Percent solutions: make 100 ml 70% EtOH from pure EtOH and Reagent grade

4. Gels: prepare 50 ml 1% agarose and pour gel

5. Microbiological media:  make 200 ml LB broth (from scratch), then make 100 ml LB agar

6. Complex buffer:  citrate (.3M) + sodium chloride (0.1M), make 100 ml

7. Pipetting:  w/ bulb, micropipette, etc:   low volume, blue, yellow, w/ plug, pipette aid/bulbs + serological.

8.  Dilution series:  volumetric and pipette skills. Going from 0.5 M to 0.1 M to 0.01, 0.001

9. Think Station

 #1

PH

 

 

Protocol

 

1.     Each group should calibrate the pH meter using the pH=7.0 and 4.0 standards.

2.     Each group should then titrate the citric acid solution supplied to them.

3.     Each group should estimate the pH of the three mystery solutions.

 

For 1.  Follow the instructions on the pH meter to calibrate the meter.

 

 

For 2.  Test the pH of the citric acid in the numbered tube. Gradually add  NaOH, by drop, checking the pH as you go, until you reach a pH of 6.5. If you overshoot 6.5 don’t panic.  Stop doing the exercise and record the pH.  If you did overshoot how would you bring the pH back down??

 

For 3.  Determine the pH of the unknowns by simply placing the probe in each of the three mystery solutions.

 

Additional:  There are pH strips and handheld pH meters also on the bench.  PLAY! 

 

#2

MOLARITY

 

1)     Materials

a)     Distilled water, NaCl, beaker, graduated cylinder, stirring rod/electric stirrer, label tape, marker, scoopula, disposable pipette, balance.

2)     Find the molar weight of the NaCl from its chemical formula

3)     Use the definition of M (molarity) to calculate the moles of salt needed to make the specified solution.

4)     Convert moles of salt needed to mass of salt

5)     Now prepare 50 ml of the 0.5 M salt solution.

a)     Put a weighing paper on the electronic balance

b)     Tare the balance

c)     Measure out the calculated mass of salt on the weighing paper

d)     Add the salt to the graduated cylinder.

e)     Add distilled water to the graduated cylinder to a bit below the required volume.  Try to wash down any salt that may have stuck to the inner wall of the graduated cylinder

f)      Mix well with stirring device.

g)     Top off the solution to the required volume mark, using level sighting to prevent parallax error, using a disposable pipette. 

6)     Label the prepared solution in a 50 ml conical tube.
#3

Percent Solutions

 

 

Materials:  Graduated cyclinder, parafilm, 95% ethanol, distilled water, pure ethanol (100%)

 

 

 

Be clear about volumes you are using.

#4

Making an agarose gel

 

Preparation and pouring of a 1% agarose gel

  1. Review the definition of a percent solution. 
  2. Calculate the grams of agarose needed in 50ml of 1X TBE to make a 1% agarose solution.
  3. Place a plastic weigh-boat on an electronic balance, and rezero the balance.
  4. Measure the amount of agarose calculated from step 1 into the weigh-boat using a clean spatula.
  5. Place the measured agarose into a clean 250ml beaker, and add distilled water until a volume of 50ml is obtained.
  6. Place the agarose solution onto a hot plate, and heat until agarose is fully dissolved.  Do not allow the agarose to boil.
  7. Remove the heated agarose solution, and swirl to insure agarose distribution.
  8. Place label tape around the open edges of an empty gel electrophoresis tray.  Firmly press the edges of the tapes onto the bottom and sides of the tray to discourage leakage.
  9. Place plastic combs in the notches at the edge of the tray.
  10. Allow the agarose solution to cool to about 60 degrees C, or until the beaker can be held in your hand.  An agarose solution above this temperature will cause leakage from the gel tray. 
  11. Pour the heated agarose solution in the tray, watching carefully to prevent leakage from around the tape.

 

NOTE:  1X TBE is an electrophoresis running buffer that is made in a 10X concentration for storage in the lab.  To make 1X TBE from the lab stock solution we simply dilute it 1:10 in distilled water.

 

 

#5

Making microbiological media

 

1       Make 200 mL of LB broth from scratch

Recipe for making LB broth = 10 g tryptone, 5 g yeast extract, 5 g NaCl for every 1 liter of broth

 

-Make ONLY 200 mL so calculate proper weights of tryptone, yeast extract and NaCl.  Place these powders in 250 mL beaker and mix with stir bar.

 

2       Use 100 mL of the above to make 100 mL of LB agar

Agar plates are 1.5% agar so calculate how much agar is needed to make 100 mL of 1.5% agar.  Add this weight of agar to proper volume of LB broth in 250 mL flask to make 100mL of LB agar.

 

Boil agar and broth solution in the microwave.

 

Pour plates using sterile technique*

 

*Sterile technique for pouring gels:  Lift several inches off plate while pouring approximately 20 mL of agar per plate, holding the cover to protect from materials falling onto the agar; immediately cover plate and allow 15 minutes to cool

 

 

 

 #6

Making a complex buffer

 

 

 

 

Complex buffer: .3 M citric acid + sodium chloride (.1 M), make 100 mL

 

Calculate grams of citric acid needed to make 0.3 M for 100 mL

Calcualte grams of sodium chloride to make 0.1 M for 100 mL

 

Measure NaCl and Citric acid with weighing boats. Add to a beaker and add approximately 80 mL of dH20. Heat and stir to dissolve. Add dH20 to reach the 100mL volume in a volumetric flask.

 

#7

Pipetting

 

Materials/Equipment:

·       Pipettes with Bulbs (Air, Suck, Empty)

·       Micropipettes

·       Transfer Pipettes

·       Plastic pipette pump

 

 

Procedures:

A.    Simple plastic pipette

·       Squeeze bulb,

·       place it into distilled water beaker

·       release the bulb to suck up

·       release bulb again to release solution drop by drop into WASTE beaker

·        

B.  Pipette with a Bulb

·       The buttons for the bulb  A(AIR), S(SUCK), and E(EMPTY)

·       Hold the A button, squeeze the bulb to create a vacuum

·       Hold S to suck-up the solution to a level higher than is necessary for the experiment           

·       Hold E to empty the solution to the desired level,(Watch the scale on you serological pipette, numbers will vary), the harder you squeeze this valve the faster the contents will flow out, go very slowly

·        

C.  Plastic pipette pump

·       Place pipette pump on the top of glass pipette

·       To suck up solution rotate dial down until the desired level is reached

·       To expel the solution rotate the dial up

·        

D.     Micropipette

·       Check the range of pipettes

·       Hold with barrel point down at all times to prevent solutions from entering the pump mechanism

·       Use correct tip for specific size of micropipette

·       Adjust volume on the micropipette by turning dial to the desired amount of solution requested

·       Depress plunger to the first level placing in solution release plunger to suck up

·       Transfer tip with solution depress plunger to first click and second click to release the last drop into one of the small brown eppendorf tubes

·       Then remove tip from the solution with plunger down and discard tip into drywaste beaker on the bench


#9

THINK STATION

Safety/Calibration/Accuracy

 

 

 

SAFETY

How do you get across the principles of safety?

Create a list of safety issues that need to be made clear to the students.

 

ACCURACY

How will you assess accuracy?

How will students apply different data collecting techniques?

 

ACCOUNTABILITY

How will you account for individual accountability as well as group accountability?

 

 

 

Techniques vary with each apparatus. (some examples follow)

 

Checking pH- cleaning probe with potassium chloride solution, color changes, using pH strips for comparison, etc.

 

Balances- calibrating with actual weights and measures, triple beam vs. electronic

Pipettes- using the proper size pipette, reading the meniscus, etc.

 

 

 

 

 

#8

Serial Dilution

 

 

MATERIALS

dH20

5 - 25ml beaker

0.5M solution NaCl

One small vial dye

25ml graduated cylinder

10 ml pipet

Labeling tape

Parafilm

 

  1. Label flasks with the following diluted concentrations: 0.1M, 0.01M, 0.001M.
  2. Put 10ml of the 0.5M NaCl solution in a 25ml beaker and add 2 drops of dye.
  3. To make 20ml of a 0.1M solution from a 0.5M solution:
    1. Using a 10ml pipet and pipettor put 4ml of the 0.5M solution containing the dye in 0.1M-labeled flask
    2.  Add 16ml dH20,measured with a 25ml graduated cylinder, and mix thoroughly
  4. To make 20ml of a 0.01M solution:
    1. Using a 10ml pipet and pipettor put 2ml of 0.1M solution into the 0.01M flask.
    2. Add 18ml dH20 to flask and mix thoroughly.
  5. To make 20ml of a .001M solution:
    1. Using a 5ml pipet and pipettor put 2ml of 0.01M solution into the 0.001M flask.
    2. Add 18ml dH20 to flask and mix thoroughly.
  6. Visually compare the 5 flasks against white, unlined paper and record your   

observations.

 

Appendix:  Standard Solutions for DNA Labs

 

 

Calcium chloride: 0.1M

 

CaCl2. 2 H2O                              14.7 g        Calcium chloride dihydrate

Water                                            100 ml

 

Dissolve the Calcium chloride in the water. Autoclave. If  you have anhydrous Calcium chloride, use 11.0 g instead.

 

 

10% Sodium dodecyl sulfate (SDS)

 

SDS                                         10 g

Water                                      90 ml

 

Dissolve the SDS in water, heating helps.  After the SDS is dissolved, adjust the final volume to 100 ml by adding a little more water.  There is no need to sterilize the solution.  DSD is also called sodium lauryl sulfate or lauryl sulfate, sodium salt.

 

Wear a dust mask when you weigh out the SDS, It is a very find powder that can irritate your nose.

 

Note that a 50% solution of dishwashing detergent can be substituted for 10% SDS in the activity Extraction of Bacterial DNA (shampoo and woolite also work.)

 

10 X TBE electrophoresis buffer

 

Tris base                                            108 g

Boric acid                               55g

0.5M  EDTA                           20 ml

water                                       to one liter

 

First, dissolve everything in slightly less than 1 liter of water, and then adjust the volume to 1 liter by adding more water.  There is no need to sterilize the solution.  If you notice that white clumps begin to precipitate in your 10X TBE, place the bottle in hot water until the clumps dissolve.  The solution can be stored at room temperature. The 10 X TBE may be autoclaved, if you like.  Some people report that autoclaving and storing in the refrigerator helps prevent precipitation.

 

To use as a buffer, dilute the 10 X stock 10-fold.  Comercially available concentrated TBE may be 5X or 20X. Dilute accordingly for 1X stock.

 

 

 

1 X TBE for Electrophoresis

 

10 X TBE                                100 ml     or     20 X TBE                 50 ml

water                                       900 ml              water                           950 ml

 

 

0.5 M  EDTA

 

EDTA, disodium salt dihydrate                     93.05 g

NaOH                                                              as described below

Water                                                              to 500 ml

 

Add 400 ml of wat4er to 93.05 g of disodium EDTA dihydrate.  While stirring the mixture, adjust the pH to 8.0 by adding NaOH (about 10.0 g of solid NaOH). EDTA will not dissolve until the pH reaches 8.0.  When the EDTA has finished dissolving, adjust the volume to 500 ml by adding water.  This solution may be autoclaved, but autoclaving is not necessary unless there is a particular reason for the solution to be sterile.

 

10 X Tris-EDTA (TE) Buffer for DNA Solutions

 

1 M Tris solution, pH 7.6 to 8.0                     10 ml

0.5 M EDTA                                                     2 ml

water                                                               88 ml

 

Autoclave the mixture.

A 10 X TE buffer is 0.1 M Tris-0.01 M EDTA.

 

1 X TE Buffer

 

10 X TE                                                          10 ml

water                                                               90 ml

 

Autoclave the mixture.

A 1 X TE buffer is 0.01 M Tris-0.001 M EDTA

 

1M Tris, pH 7.6 to 8.0

 

Tris base                                                         12.1 g

Concentrated HCL                                        as described below

Water                                                              to 100 ml

 

Dissolve the Tris base in about 80 ml of water in a beaker.  Add concentrated HCl and stir until the pH is in the 7.6 - 8.0 range.  It is best to add the HCl and stir it in a chemical hood because of fumes from the acid.  The solution will become hot.  After it cools put the solution in a graduated cylinder and bring the volume to 100 ml with water.