Biotechology

Skill I: Using Micropipets, Transfer Pipets, and a Balance (Time: 15 minutes)

1. Enter the room in PEE (Gloves, Goggles, Closed-toe toe shoes)

2. Label 3 centrifuge tubes as stated on paper

3. Weigh the empty tubes and record mass on paper

4. Using the 20-200 ul micopipet

   1. Pipette 200 ul into the tube, label 200p

   2. Pipette another 200 ul into the tube labeled 200p

   3. Pipette 100 ul into the tube labeled 200p

5. Using the 100-1000 ul mircopipet

   1. Pipette 500 ul into 1000p tube, close tube

6. Using the transfer pipet

   1. Transfer 500 ul into TP tube, close tube

7. Calculating weight

   1. Weigh all three tubes again, then subtract empty tube weight from weight of tube with liquid for total mass in grams

   2. Record the mass in grams on paper

8. Cleaning Up

   1. Throw away tubes, pipet, and Transfer pipet

   2. Clean work area

   3. Removed PPE

   4. Wash hand/ alcohol wipes

Skill II: Restriction Digestion Reaction (Time: 15 min)

1. Enter room in PPE

2. Label 6 tubes based on the DNA sample on paper

3. Pipet 10 ul of each sample into their tubes

4. Pipet 10 ul of ENZ into each tube, then mixed by pipetting up and down

5. Cape tube and mix by flicking'

6. Pulse-spin in centrifuge or tap on table gently

7. Say, “Incubating over night at room temperature”

8. Cleaning up

Skill III: DNA Gel Electophoresis (20 minutes)

1. Enter room in PPE

2. Preparing Samples

   1. Collect liquid at the bottom for 6 tubes, can centrifuge them in pulse-spin for 5-10 seconds or gently tap on table

   2. Pipet 5 SLB(sample buffer) into each tube, mix by pipetting up and down

   3. Loading Gel

      1. Place precast gel in chamber facing toward black

      2. Put in TAE until all wells are covered

      3. Pipet 10 ul of standard in first well

      4. Pipet 20 of each sample into well

      5. Record what wells each sample is in

      6. Placed lid on chamber and plug wires

      7. Turn on power and 100V or say it

      8. Say, “Run for 30 minutes”

      9. Say, “run completed”, turn off power and remove lid

      10. Clean up

          
          

Skill IV: DNA Gel Interpretation (15 minutes)

1. Use ruler to measure distance (mm) that each fragment/band traveled from well, record each on paper

2. Use semilog paper to plot the distance verus size

3. Draw a line of best fit through points

4. Use graph to estimate the fragment zie for each band in the samples, record estimates on table

Skill V: Bradford Protein Quantitation Assay (20 min)

1. Enter in PPE

2. Label two tubes, A and B

3. pipet 2 ul of sample A in to tube A

4. Pipet 98 ul of PBS into tube A

5. pipet 2 ul of sample B into B

6. pipet 98 ul of PBS into B

7. Mix by flicking

8. Label two curv A and B

9. Pipet 20 ul into each curve

10. Preparing standards

    1. Label 8 curvs, Blank,0.125, 0.250, 0.500, 0.750, 1.000,1.500,2.000

    2. Pipet 20 ul of PBS into blank

    3. pipet 20 of each protein into other curves

    4. 1000 ul of bard ford into each curve, mix by pipetting up and down

    5. Say, “curvettes incubate at room temperature for 5 minutes”

    6. Compare the sample curves with protein ones, estimate and say it out loud

    7. Clean up

Skill VI: Bacterial Transformation ( 20 minutes)

1. Enter room in PPE

2. Preparing for heat shock

   1. Label one tube +pGLO and another -pGLO

   2. Pipet 250 ul of CACI into each tube, place on ice

   3. Use sterial look to scrape E.clio off plate

   4. Transfer to +pGLO tuber and swril, then place back on ice

   5. Throw loop away

   6. Use loop to get eclio

   7. Transfer to -pGLO tuber and swirl

   8. Throw loop

   9. pipet 10 ul of plasmid into +pGLO

   10. Throw tip away

   11. Place both tubes in ice

   12. Get 4 algar plates label them

       1. LB/amp +pGLO

       2. LB/amp/ara +pGLO

       3. LB/amp -pGLO

       4. LB -pGLO

   13. Say, “ 10 minute incubation on ice completed”

   14. Transfer tubes from ice into 42 degree water bath for 50 seconds, then immediately place tubes on ice

   15. Say,”tubes remain on ice for 2 minutes”

   16. Remove tubes from ice and pipet 20 ul of LB into each tube

   17. Throw tip

   18. Say,”Samples incubated at room temperature for 10 min

   19. Platting Bacteria

       1. Mix tubes by flicking

       2. Pipet 100 ul of mixture onto either + or - plate

       3. use loop to spread bacteria

       4. Stacked plate downwards

       5. Say, “plate incubates at 37 celusi for 16-24 hours”

   20. Clean up

Skill VII: Calculation of transformation efficiency

1. Enter in PPE

2. Count the number of colonies on the plate

3. Calculate how much DNA was spread on plate

   1. Total amount of DNA started with

      1. Concentration x Volume of DNA

         1. ul divide away into ug

   2. What fraction was spread

      1. Volume of solution spread on plate/ Volume in the tube

   3. Total ug of DNA in tube x fraction of tube used

4. Calculate Transformation efficiency:

   1. Number of colonies/ amount of DNA used

5. Remove PPE

Skill VIII: Qualitative ELISA (20)

1. Enter in PPE

2. Label 12 strips

   1. Three +

   2. Three -

   3. Three S

3. Antigen Incubation

   1. Transfer 50 ul of AG into each well

   2. incubate at room temp for 2 minutes

   3. Follow wash protocol:

      1. Tip stip up-side down onto stack of paper and gentle tap a few times

      2. Throw paper away

      3. Use transfer pipette fill each well with wash buffer

      4. Tip stip up-side down onto stack of paper and gentle tap a few times

   4. Sample incubation

      1. Transfer 50 ul of the positive + into three wells

      2. Transfer 50 ul of the negative - into three wells

      3. Transfer 50 of the sample into three wells

      4. Incubate at room temp for 2 min

      5. Wash protocol (2 times)

      6. Transfer 50 ul of ELA

      7. incubate samples for 2 minutes

      8. Wash protocol 3 times

      9. 50 ul of SUB

   5. Clean up