Aliquoting Guide for Lab 6

We have provided sufficient reagents for the number of students you indicated on the Materials Request form, including a bit extra for pipetting errors. All solutions should be aliquoted into the provided 1.5 mL microcentrifuge tubes, unless otherwise indicated.

See teacher instructions for Lab 6 on methods for growing the culture of transformed cells. We recommend doing this step with the commercially ligated/purified plasmid used in the short series (pARA-R). The sterile broth provided has been pre-calculated so there is enough for each group to get the 2mL required. 

NOTE: You must grow the culture at least 24 hours to get good expression of the rfp before you can begin Lab 6.

Day 1. Lysing of cells from the overnight mFP expression.

Label tube Contents of tube Aliquot Actually used
EC overnight E. coli culture 1mL**** 2mL
LyB Lysis buffer 160µL 150µL
EB Elution buffer 160µL 150µL

****You will need to do this twice into the same tube, once before and once following centrifugation after the supernatant has been discarded.


Day 2. Purification of RFP using column chromatography

Size of tube Label tube Contents of tube Aliquot Actually used
15 mL BB

Binding buffer 
(4M (NH4)2SO4)

250µL 200µL
15 mL CEB

Column equilibration buffer
(2M (NH4)2SO4)

3.5mL 3mL
15 mL WB

Wash buffer 
(1.3M (NH4)2SO4)

1.5mL 1mL
15 mL EB

Elution buffer
(10mM TE)

2.5mL 2mL


Be certain that the last group of students flush the columns with 2 mL of equilibration buffer. Allow the equilibration buffer to drain but leave about 0.5cm of it above the resin bed. Be certain that all tubes are capped, that the extensions have been removed and small yellow caps replaced, and check to see that the stop cocks are in the off position before storing for the next school or class. Always store the columns UPRIGHT. Return the columns to the plastic, lidded box.

 

 

Resource category
ABE Lab Specific Resources
Related Lab Lab 6: Obtaining the Protein