User:DavidRamos/Lab/2006-6-14

Morning
 Plasmid Miniprep  Nanodrop results -1: 31.2ng/uL 260/280:1.75 260/230:1.92 -2: 38.5ng/uL 260/280:1.83 260/230:1.87 -3: 33.8ng/uL 260/280:1.70 260/230:1.44
 * lac operon promoter
 * R0010
 * promoter and GFP
 * E0241
 * GFP
 * E7104
 * DNA Miniprep of transformant colonies
 * Out of 5mL of liquid culture, reserved 1mL for gylcerol+freeze and 4mL other
 * followed QIAprep Miniprep Kit for Microcentrifuge directions
 * Eluted with warm dH20
 * Put at 40C for ~2 min to evaporate ethanol before elution
 * Forgot to label after elution --> don't know what is what
 * Sol'n: During digest will have to run PCR, can tell R0010 from rest, but E7104/E0241 is only different by 30bp; if doesn't work can flip and try two experiments.
 * Nanodrop demonstration

PROBLEM: Messed up labeling of the plasmids! To diagnose, ran a 15min e-Gel to find out which is R0010.


 * Digestion of vector/insert
 * Digested R0010 (200bp cutout) as vector at S and P site.
 * .5uL Spe1, .5uL Pst1
 * 11uL h20
 * 2.5uL 10X BSA
 * 2.5uL #2 NebBuffer
 * 8uL DNA
 * Digested other 2 (~900bp cutout) as insert at X and P site.
 * .5uL Xba1, .5uL Pst1
 * 11uL h20
 * 2.5uL 10X BSA
 * 2.5uL #3 NebBuffer
 * 8uL DNA
 * Incubate @ 37C for 1h
 * Phosphatase
 * 80C@15min to kill enzyme activity
 * Used CIP (1 unit) into the R0010, 1h@37C
 * Run on 1% agarose gel
 * Image, Cutout, and Purify
 * Can isolate the three from the gel

Result

Ladder=1kb+ Lane 1=R0010 (#1) Lane 2=E0241 (#2) Lane 3=E7104 (#3)