June 14 (Wednesday): Difference between revisions
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- checked www.neb.com to determine correct Buffer | - checked www.neb.com to determine correct Buffer | ||
- digested @ 37 dC for 1 hr | - digested @ 37 dC for 1 hr | ||
- heat shocked @ 97 dc for 15 min to inactivate restriction enzymes | |||
===Glycerol Stock=== | ===Glycerol Stock=== |
Revision as of 12:14, 14 June 2006
Miniprep
1. Plasmids (2 samples each)
R0010: lac operon promoter E7104: T7 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
Digestion
1. Materials
8 microL DNA (R0010, E0241) 2.5 microL 10x BSA 2.5 microL 10x Buffer (Buffer 2) 11 microL dH2O 0.5 microL enzyme 1 and 2 (SpeI and PstI, XbaI and PstI) total vol = 25 microL
2. Protocol
- checked www.neb.com to determine correct Buffer - digested @ 37 dC for 1 hr - heat shocked @ 97 dc for 15 min to inactivate restriction enzymes