Minnesota/9 July 2008

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1. Single Digests: Performed today. Incubated digested samples for 2hrs @ 37C. Refer to table below.

Gene	10x Buffer	BSA Protein	H20	DNA	RE
Promoter	5.0uL	0.5uL	40.4uL	2.1uL	2.0uL, SPE
LAMBDAcI	5.0uL	0.5uL	29.5uL	15.0uL	1.0uL, XBA 1
LAMBDAcI	5.0uL	0.5uL	29.5uL	15.0uL	1.0uL, SPE
Terminator	5.0uL	0.5uL	38.0uL	4.5uL	2.0uL, XBA 1

NOTE: RE = Restriction Enzyme

2. Ligation: Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table

below.

Genes	10x Buffer	H20	Base Vector	Insert	DNA Ligase
LAMBDAcI/Terminator	2.0uL	10.7uL	2.0uL (Term.)	4.3uL (LAMBDAcI)	1.0uL
Promoter/LAMBDAcI	2.0uL	10.7uL	2.0uL (Pro.)	4.3uL (LAMBDAcI)	1.0uL

3. Double Digests: Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight.

Gene	10x Buffer	BSA Protein	H20	DNA	RE 1	RE 2
Promoter/LAMBDAcI	5.0uL	0.5uL	22.5uL	20.0uL	1.0uL, EcorI	1.0uL, PstI
LAMBDAcI/Terminator	5.0uL	0.5uL	20.0uL	20.0uL	1.0uL, EcorI	1.0uL, PstI
Base Vector	5.0uL	0.5uL	15.0uL	15.0uL	1.0uL, EcorI	1.0uL, PstI

NOTE: RE = Restriction Enzyme

4. Make/Pour Ampicillin Plates

5. Order Primers for Part Sequencing

6. Purified Plasmid Prep: Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.