User:Eric Ma/Notebook/MICB323 Lab Book/2009/02/10

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Part 1: Agarose Gel of Plasmid DNA from Previous Lab

 * Conditions Used:
 * 100 mL of 0.7% agarose gel + 10µL SYBR Safe Stain

Summary of Tube Labels

 * EM1-4 EcoRI - Manipulation of EcoRI concentration for digestion of isolated pDNA
 * EM1-4 NaCl - Manipulation of NaCl concentration for digestion of isolated pDNA
 * EM#1-2 - Isolated plasmid DNA from CaCl2 (#1: transformed) and (#2: untransformed)
 * EM1-3 - Digested plasmid DNA from CaCl2 (#1: transformed), (#2: untransformed),(#3: Sunny's Part 2)

Lanes on the gel and volumes added to the wells
Gel was run for 1 hour @ 120 volts. Noticed that the samples in lanes 3, 5 and 7 had a tendency to float off.
 * 1) Supercoiled Ladder (10µL)
 * 2) 380 and 1000 standard (10µL)
 * 3) EM1 - Untransformed and digested pDNA from CaCl2 (10 µL Sample + 2µL 6X Loading Buffer)
 * 4) EM#1 - Isolated untransformed pDNA from CaCl2 (10 µL Sample + 2µL 6X Loading Buffer)
 * 5) EM2 - Transformed and digested pDNA from CaCl2 (10 µL Sample + 2µL 6X Loading Buffer)
 * 6) EM#2 - Isolated Transformed pDNA from CaCl2 (10 µL Sample + 2µL 6X Loading Buffer)
 * 7) EM3 - Digested p421 plasmid DNA inserted via CaCl2 (10 µL Sample + 2µL 6X Loading Buffer) (Note: From Sunny)
 * 8) EM#3 - Undigested p421 plasmid DNA inserted via CaCl2 (10 µL Sample + 2µL 6X Loading Buffer) (Note: From Sunny)
 * 9) Ligation Reaction (8µL including 1.5µL of 6X Loading Buffer)
 * 10) Mass Ruler (10 µL)
 * 11) EM1 EcoRI (18 µL)
 * 12) EM2 EcoRI (18 µL)
 * 13) EM3 EcoRI (18 µL)
 * 14) EM4 EcoRI (18 µL)
 * 15) EM1 NaCl (18 µL)
 * 16) EM2 NaCl (18 µL)
 * 17) EM3 NaCl (18 µL)
 * 18) EM4 NaCl (18 µL)
 * 19) Mass Ruler (10 µL)

Problem: SDS Added to Recombinant Viral Lysates

 * Mark added SDS to the recombinant viral lysates by accident.
 * This would cause problems - volumes are wrong, and all the protein would be denatured.
 * 50 µL + 200 µL = 250 µL total volume. The lysate is 4/5 as concentrated as it is supposed to be.
 * Unable to do BSA Assay
 * Hence, I did the BCA Assay along with Mark, using the (need to find out - did we use the recombinant or wild-type virus?) virus.
 * See lab manual for changes made.
 * We did a 1/3 dilution of each viral lysate rather than a 1/4 dilution, putting in 40µL of viral lysate and 80µL of sterile distilled H2O.

BCA Assay Plate Results

 * Please see [[Media:090210_BCA_Assay_Results.xlsx|this spreadsheet]] for details on calculations.

Eric's Plate (Recombinant Virus) [NOTE: DO NOT USE THE RESULTS FROM ERIC'S PLATE]

 * Guide to plate:
 * Standard Curve
 * Calculated Concentrations

Sunny's Plate (Wild-Type Virus)

 * Guide to plate:
 * Standard Curve:
 * Calculated Concentrations:

BSA Assay Results

 * Notes: Done up by Crystal




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