Minnesota/14 July 2008

From 2008.igem.org

(Difference between revisions)

Revision as of 17:02, 14 July 2008

Back to Notebook Home
Go to Previous Day (July 11)	Go to Next Day (July 15)

1. Plasmid DNA Purification Using the QIAprep: Use 2mL LB cultures of ligation products from 07-11-2008. Procedure:

a. Resuspend bacterial cells in 250uL Buffer P1 and transfer to microcentrifuge tube

b. Add 250 uL Buffer P2 and invert 4-6 times

c. Add 350uL Buffer N3 and mix immediately by inverting 4-6 times

d. Centrifuge for 10 minutes @ 13,000 rpm in a table-top centrifuge. White pellet will form (made up of cell debris).

e. Apply supernatants from step D to the QIA prep spin columns. Centrifuge for 30-60 seconds --> discard the flow through.

f. Wash the QIA prep spin column by adding 0.5mL (500uL) Buffer PB. Centrifuge for 30-60 seconds. Discard the flow through.

g. Wash QIA prep spin column by adding 0.75mL (750uL) Buffer PE. Centrifuge for 30-60 seconds. Discard the flow through.

h. Centrifuge for additional 1 minute to remove residual buffer.

i. Place the QIA prep spin column in a clean 1.5mL centrifuge tube. To elute DNA, add 50uL of Buffer EB (10mM Tris-Cl, pH 8.5) to center of spin column and centrifuge.

NOTE: Used Buffers to purify or washout any cell debris so only has DNA. Used spin column b/c the column binds to DNA (has DNA affinity) and all other solution will drain through.

2. Spectrophotometry: 'Spec' purified preps to check concentration of DNA in the ligated products. Refer Spectrophotometry Results link on Notebook page.

3. Double Digest: Perform double digest. Incubate @ 37C for 2-20hrs. Heat inactivate enzyme @ 65C for 15 mins. Vector Dephosphorylation next; Base Vector only.

GFP + Terminator => GFP:Term.

Pro/LAMBDAcI + Terminator => Pro:LAMBDAcI:Term.

Pro + LAMBDAcI/Terminator => Pro:LAMBDAcI:Term.

Refer to Double Digest table below.

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2
GFP (1250 ng/uL)	5.0uL	0.5uL	41.5uL	1.0uL	1.0uL, EcoRI	1.0uL, Spe1
Terminator	5.0uL	0.5uL	17.5uL	25.0uL	1.0uL, Xba1	1.0uL, Pst1
Promoter/LAMBDAcI	5.0uL	0.5uL	40.5uL	2.0uL (L3b)	1.0uL, Pst1	1.0uL, Spe1
Promoter/LAMBDAcI	5.0uL	0.5uL	32.5uL	10.0uL (L3c)	1.0uL, Pst1	1.0uL, Spe1
LAMBDAcI/Terminator	5.0uL	0.5uL	27.1uL	15.4uL (L4b)	1.0uL, EcoRI	1.0uL, Xba1
LAMBDAcI/Terminator	5.0uL	0.5uL	20.3uL	22.2uL (L4c)	1.0uL, EcoRI	1.0uL, Xba1
BaseVector (35 ng/uL)	5.0uL	0.5uL	13.5uL	29.0uL	1.0uL, EcoRI	1.0uL, Pst1
TetR Promoter	5.0uL	0.5uL	8.5uL	34.0uL	1.0, EcoRI	1.0uL, Spe1

@@ Line 32: / Line 32: @@
 |'''2. Spectrophotometry:''' 'Spec' purified preps to check concentration of DNA in the ligated products. Refer Spectrophotometry Results link on Notebook page.
 |-
-|'''3. Double Digest:'''
+|'''3. Double Digest:''' Perform double digest. Incubate @ 37C for 2-20hrs. Heat inactivate enzyme @ 65C for 15 mins. Vector Dephosphorylation next; Base Vector only.
 |-
 |GFP + Terminator => GFP:Term.