Amgen Lab Study Guide

Amgen Lab Study Guide

Micropipettor & Centrifuge

1a) What the window look like on a P-20 if you dialed in 6.3 µL?

1b) What the window look like on a P-200 if you dialed in 63 µL?

1c) What the window look like on a P-1000 if you dialed in 630 µL?

2a) What is the purpose of the “first stop” on the micropipettor?

2b) What is the purpose of the “second stop?”

3a) If you were using a microfuge with 6 openings and you needed to spin down 2 tubes, how would you arrange the tubes? *For any of these centrifuge questions, indicate if you need to use 1 “blank” tube.

3b) If you were using a microfuge with 6 openings and you needed to spin down 3 tubes, how would you arrange the tubes?

3c) If you were using a microfuge with 6 openings and you needed to spin down 4 tubes, how would you arrange the tubes?

3d) If you were using a microfuge with 6 openings and you needed to spin down 5 tubes, how would you arrange the tubes?

LAB 2a

1) How would you identify a Restriction Site?

2) How many restriction sites are on the pARA-R plasmid?

3) What do the restriction enzymes, Bam H I and Hin d III, do to the plasmid?

4) How are the R+ and R- tubes different?

5) Why did you have to add distilled water to the R- tube?

6) What was happening in the 37ºC water bath?

Lab 4a- General Gel Electrophoresis Questions

1) What are 2 purposes of using loading dye?

2) Gel electrophoresis separates molecules based on what 2 properties? (circle which property used in Lab 4a)

3) What is the purpose of “marker”/DNA ladder?

4) What is the purpose of soaking the gel in the stain EtBr (Ethidium Bromide) & then rinsing it in distilled water?

5) Why was DNA loaded into the negative end of the gel electrophoresis box?

Lab 4a- Gel Electrophoresis Questions Specific to our Lab

6) Draw out the banding pattern found in the R+ and R- lanes. Be sure to label both the plasmid form names and fragment size for each fragment.

7) Draw in a 3rd lane labeled “X.” Show what an incomplete digest would look like. (Digestion has started but it hasn’t finished.)

Lab 5

1) How are the P+ and P- tubes different?

2) What is the purpose of “heat-shock?”

3) What does it mean that the E. coli bacteria is “competent” (aka: it is a competent cell).

4) What substance is the “food” for the bacteria?What 2 forms was it found in for the lab?

5) Draw and label the pARA-R plasmid. Also, describe the function of the following areas:

-rfp

-PBAD

-araC

ampr

6) Explain the relationship between PBAD, araC protein, arabinose, and transcription of rfp.

7) What is a bacterial colony?

8) Why did E. coli grow on both the P- and P+ sides of the LB plate?

9) Why did E. coli only grow on the P+ side of the LB/amp plate?

10) Why are the colonies red on the LB/amp/ara plate but not on the LB/amp plate?

11) What is the formula used to calculate transformation efficiency? (include units)

12) If plate#1 was spread with 0.065 g and had 200 colony forming units on it, and plate#2 was spread with 0.085 g and had 250 colony forming units on it, which one has higher transformation efficiency? EXPLAIN.Don’t forget to use scientific notation.

Lab 6 Preparing an Overnight Culture of E. Coli

1) Why was ampicillin included in the overnight culture?

Lab 6 Purification of mFP from an Overnight Culture

1) What was the purpose of adding lysis buffer to the cell culture?

2) What property of the protein was used to purify out the protein in the chromatography columns?

3) What type of resin was used in the chromatography column?

4) Why is mFP placed in a high salt concentration?

5) What is the purpose of the wash buffer?

6) What is the purpose of running a solution of very low salt concentration through the chromatography column?