Amgen Biotech Experience Alternate Aliquoting Guide Lab 1 Some tools of the trade



Download 25.4 Kb.
Date04.06.2018
Size25.4 Kb.
Amgen Biotech Experience

Alternate Aliquoting Guide

Lab 1 Some tools of the trade

Size of Tube

Label tube

Contents of tube

*Aliquot

Actually used

1.5 mL

RD

Red practice solution




Varies

1.5 mL

S1

Solution 1 - Blue and purple dye




10uL

1.5 mL

S2

Solution 2 - Blue, purple, yellow dye




10uL

1.5 mL

S3

Solution 3 – Purple dye




12uL

1.5 mL

dH2O

**Distilled water




varies

*A class set (12 tubes each) has been pre-aliquoted and can be found in one of the equipment boxes in a plastic microfuge tube box. When these solutions get low, please inform your site coordinator. ** dH2O is used for several labs.

Lab 2 Preparing to clone the RFP gene: digesting the pKAN-R and pARA plasmids

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

A

pARA (80 ng/uL)

10uL

8uL

1.5 mL

K

pKAN-R (80 ng/uL)

10uL

8uL

1.5 mL

RE

BamH I and Hind III

5uL

4uL

1.5 mL

2.5xB

2.5x restriction buffer

20uL

16uL

1.5 mL

dH2O

Distilled water

*

4uL



Lab 2A Preparing to verify the RFP gene: digesting the pARA-R plasmid

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

pR

*pARA-R (70 ng/uL)

10uL

8uL

1.5 mL

RE

BamH I and Hind III

3uL

2uL

1.5 mL

2.5xB

2.5x restriction buffer

12uL

8uL

1.5 mL

dH2O

Distilled water

*

2uL

*There are two different tubes of pARA-R for the Abridged Genetic Engineering Sequence, Lab 2A uses the 70 ng/uL pARA-R. Check the reagent tube labels carefully.

Lab 3 Building the pARA-R plasmid

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

5xB

5x ligation buffer

4uL

3uL

1.5 mL

LIG

T4 DNA ligase

2uL

2uL

1.5 mL

dH2O

Distilled water

*

2uL



Lab 4 Verification of restriction and ligation using gel electrophoresis

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

DL

*1 kb DNA ladder

10uL

10uL

1.5 mL

LD

**Loading dye

20uL

10uL

*The DNA ladder contains loading dye and is easily mistaken for loading dye – warn your students to check labels on tubes carefully. If you run short of loading dye, you may substitute Solution 2 from Lab 1, it is the same as loading dye.

Lab 4A Verification of the recombinant plasmid using gel electrophoresis

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

DL

*1 kb DNA ladder

10uL

10uL

1.5 mL

LD

**Loading dye

10uL

6uL

*The DNA ladder contains loading dye and is easily mistaken for loading dye – warn your students to check labels on tubes carefully. If you run short of loading dye, you may substitute Solution 2 from Lab 1, it is the same as loading dye.

Lab 5 Transforming bacteria with the ligation products

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

LB

Luria broth

350uL

300uL

1.5 mL

CC

*Competent cells

100uL

100uL

*Do not aliquot the competent cells until class time, 15 minutes before students begin the lab.

Lab 5A Transforming bacteria with recombinant plasmids

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

LB

Luria broth

350uL

300uL

1.5 mL

RP

**pARA-R (10 ng/uL)

12uL

10uL

1.5 mL

CC

*Competent cells

100uL

100uL

*Do not aliquot the competent cells until class time, 15 minutes before students begin the lab. **There are two different tubes of pARA-R for the Abridged Genetic Engineering Sequence, Lab 5A uses the 10 ng/uL pARA-R. Check the reagent tube labels carefully.

Lab 5B Transforming bacteria with recombinant plasmids

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

LB

Luria broth

350uL

300uL

1.5 mL

RP

pARA-R (10 ng/uL)

12uL

10uL

1.5 mL

CC

*Competent cells

100uL

100uL

*Do not aliquot the competent cells until class time, 15 minutes before students begin the lab

Lab 6 Purifying the fluorescent protein

Part A

Size of tube

Label tube

Contents of tube

Aliquot

Actually used

1.5 mL

EC

LB/amp/ara culture of E. coli

*2 x 1mL

2mL

1.5 mL

LyB

Lysis buffer

160uL

150uL

1.5 mL

EB

Elution buffer

200uL

150uL

*Each group will centrifuge a total of 2mL of the LB/amp/ara culture, but they will have to receive 2 aliquots of 1mL as 2mL will not fit into a 1.5mL microfuge tube.

Part B

*Size of tube

Label tube

Contents of tube

*Aliquot

Actually used

15mL

BB

Binding buffer 4M (NH4)2SO4




200uL

15ml

WB

Wash buffer 1.3M (NH4)2SO4




1mL

15mL

EB

Elution buffer 10mM TE




2mL

15mL

CEB

Column equilibration buffer 2M (NH4)2SO4




2mL

*You will be provided with either sets of 15mL tubes of each buffer or one large container of each buffer. If you have the 15mL tubes, one can be given to each group. If you have the larger container, divide each buffer into a set of flasks that can be shared by two groups.

10-1-2013




Share with your friends:


The database is protected by copyright ©dentisty.org 2019
send message

    Main page