June 14 (Wednesday): Difference between revisions
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==Miniprep== | ===Miniprep=== | ||
1. Plasmids (2 samples each) | 1. Plasmids (2 samples each) | ||
R0010: lac operon promoter | R0010: lac operon promoter | ||
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==Digestion== | ===Digestion=== | ||
Revision as of 08:42, 14 June 2006
Miniprep
1. Plasmids (2 samples each)
R0010: lac operon promoter E7104: promoter + GFP E0241: GFP
2. Protocol
- w/ 5 mL samples, 1 mL saved for glycerol stock
- rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet
- supernatant removed and pellet resuspended in 250 microL Buffer P1
- 250 microL Buffer P2 added and tube inverted 4-6 times for mixing
(no vortexing to prevent shearing of genomic DNA; let sit for no longer than 5 min)
- 350 microL Buffer N3 added and tube inverted 4-6 times for mixing
- centrifuged @ 13,000 rpm for 10 min
(formation of white pellet)
- supernatant transferred to QIAprep spin column and centrifuged for 1 min
- 0.5 mL Buffer PB added and centrifuged for 1 min
(optional)
- added 0.75 mL Buffer PE for washing and centrifuged for 1 min
- flowthrough discarded and centrifuged for 1 min to remove residual buffer
- QIAprep column transferred to 1.5 eppendorfs
- 50 microL water added to center of column
(let column stand for 1 min)
- centrifuged for 1 min
- nanodropped
