1 GENERAL BIOLOGY LAB 1 (BSC1010L) Lab #10: Biotechnology ...

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Develop a Hypothesis:Based on your expectations (Table 1), state null and alternative hypotheses regarding what youexpect to see if the bacteria plated on an ampicillin rich medium were successfully transformed.Procedure:1. Obtain two sterile Eppendorf tubes.a. Mark one tube “+ DNA” – this tube will receive the plasmid.b. Mark the other “- DNA”.2. On the side of both tubes, write your group number.3. Add 250µL of ice-cold calcium chloride (CaCl 2 ) to each tube using a sterile transferpipette. Addition of CaCl 2 to the host cells, followed by heat shock (see step 14),increases the likelihood of plasmid uptake. Cells that are able to take up the plasmidDNA are referred to as competent cells.4. Place both tubes on ice.5. Using one side of a plastic sterile loop, transfer a cell mass of E. coli cells from the starterplate into the + DNA tube by sweeping the cells superficially.a. Note: You only need a small quantity of bacteria. Do NOT completely fill theloop.Notes:• Be careful not to transfer any agar from the plate along with the cell mass• Immerse the cells on the loop in the calcium chloride solution in the + DNA tubeand vigorously tap the loop against the wall of the tube to dislodge the cell mass.Hold the tube up to the light to observe that the cell mass has fallen off the loop.6. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transferpipette. Be careful not to spill the contents.• Examine the tube against the light to confirm that no visible clumps of cellsremain in the tube or are lost in the bulb of the transfer pipette. The + DNAtube should be appear cloudy while the - DNA tube should be clear (Fig. 1).4
Figure 17. Return the + DNA tube to ice.8. Using the opposite end of the plastic sterile loop, transfer a mass of cells to the - DNAtube and suspend as described in steps 5 and 6 above.9. Return the - DNA tube to ice. Both tubes should now be on ice.10. Your instructor will add 10µL of plasmid DNA (concentration = 0.005 µg/µl) to the+ DNA tube. Gently tap the tube with your fingers to mix the DNA with the cells. Tokeep consistency between the two treatments, gently mix the – DNA tube as well.11. Centrifuge both tubes in a microcentrifuge for approximately 5 seconds before returningboth tubes to ice. BE SURE TO NOTE THE SLOT NUMBERS WHERE YOUPLACED YOUR GROUP’S TUBES SO YOU DON'T GET THEM CONFUSEDWITH THOSE OF ANOTHER GROUP. ALSO MAKE SURE THAT THE TUBESARE BALANCED INSIDE THE CENTRIFUGE BEFORE SPINNING (Fig. 2). Ifyou are unsure about this step, speak to your instructor before continuing.12. Incubate the tubes on ice for 30 min.Figure 25
Page 3: Table 1:LB -Condition Expected resu
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Page 9 and 10: A. B.Figure 6. Plates illustrating
Page 11 and 12: c. Since only a portion of the + DN
Page 13: 5. Can you think of a case where th

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