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

  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)

Gel was run for 1 hour @ 120 volts. Noticed that the samples in lanes 3, 5 and 7 had a tendency to float off.

Part 3: Determination of Viral Lysate Protein Concentrations using the Bradford and Bicinchoninic Acid Protein Assays

Notes

  1. Crystal and Mark did the spinning + extraction of lysates.
  2. Sunny and I will do the recombinant virus samples; Mark and Crystal will do the wild-type virus samples.
    1. Sunny does BCA assay. (This did not happen.)
    2. I do BSA assay. (This did not happen. See below)

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.

Results

Gel Electrophoresis

File:090210 Gel eric ma.tif

Southern Hybridization

File:090303 Southern Image.tif

BCA Assay Plate Results

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

Row\\Column 1 2 3 4 5 6 7 8 9 10 11 12
A 0.104 0.1 0.102 0.098 0.097 0.106 0.839 0.841 0.814 0.892 0.881 0.918
B 0.105 0.105 0.101 0.1 0.101 0.109 2.894 2.813 2.819 2.812 0.035 0.034
C 0.173 0.167 0.164 0.163 0.159 0.161 2.835 2.788 2.847 2.801 0.034 0.034
D 0.227 0.23 0.217 0.209 0.208 0.215 2.746 2.746 2.785 2.747 0.034 0.035
E 0.359 0.343 0.336 0.33 0.319 0.326 2.73 2.786 2.728 2.688 0.034 0.034
F 0.457 0.446 0.492 0.453 0.437 0.426 2.677 2.667 2.675 2.669 0.035 0.033
G 0.544 0.539 0.533 0.542 0.539 0.529 2.758 2.734 2.729 2.714 0.034 0.034
H 0.703 0.686 0.723 0.754 0.754 0.778 0.034 0.034 0.034 0.035 0.034 0.034
  • Guide to plate:
Wells Sample
A1-A6 0 µg/mL
B1-B6 25 µg/mL
C1-C6 125 µg/mL
D1-D6 250 µg/mL
E1-E6 500 µg/mL
F1-F6 750 µg/mL
G1-G6 1000 µg/mL
H1-H6 1500 µg/mL
A7-A12 2000 µg/mL
B7-B12 2.5 hr (-) virus
C7-C12 2.5 hr (+) virus
D7-D12 2nd harvest + virus
E7-E12 3rd harvest + virus
F7-F12 4th harvest + virus
G7-G12 5th harvest + virus
H7-H12 N/A
  • Standard Curve

BCA Assay Standard Curve (Eric's Plate)

  • Calculated Concentrations
Sample Calculated Concentration (µg/mL)
0 µg/mL -49.6
25 µg/mL -43.7
125 µg/mL 111.2
250 µg/mL 246.1
500 µg/mL 545.2
750 µg/mL 840.4
1000 µg/mL 1058.3
1500 µg/mL 1554.1
2000 µg/mL 1887.0
2.5 hr (-) virus 6887.8
2.5 hr (+) virus 6845.3
2nd harvest + virus 6688.6
3rd harvest + virus 6630.2
4th harvest + virus 6475.4
5th harvest + virus 6632.1

Sunny's Plate (Wild-Type Virus)

Row\\Column 1 2 3 4 5 6 7 8 9 10 11 12
A 0.101 0.111 0.098 0.099 0.105 0.1 0.737 0.795 0.791 0.731 0.79 0.752
B 0.101 0.097 0.1 0.091 0.098 0.098 0.034 0.034 0.034 0.034 0.035 0.034
C 0.158 0.153 0.152 0.154 0.144 0.158 0.335 0.236 0.204 0.211 0.04 0.034
D 0.212 0.21 0.209 0.205 0.197 0.212 0.384 0.257 0.231 0.251 0.034 0.034
E 0.334 0.299 0.287 0.287 0.287 0.266 0.624 0.446 0.365 0.65 0.034 0.034
F 0.409 0.42 0.404 0.395 0.398 0.388 0.516 0.331 0.292 0.367 0.033 0.033
G 0.469 0.488 0.41 0.429 0.476 0.483 0.577 0.72 0.313 0.297 0.034 0.034
H 0.684 0.708 0.622 0.591 0.609 0.642 0.415 0.325 0.265 0.267 0.034 0.034
  • Guide to plate:
Wells Sample
A1-A6 0 µg/mL
B1-B6 25 µg/mL
C1-C6 125 µg/mL
D1-D6 250 µg/mL
E1-E6 500 µg/mL
F1-F6 750 µg/mL
G1-G6 1000 µg/mL
H1-H6 1500 µg/mL
A7-A12 2000 µg/mL
B7-B12 N/A
C7-C12 2.5 hr (-) virus
D7-D12 2.5 hr (+) virus
E7-E12 2nd harvest + virus
F7-F12 3rd harvest + virus
G7-G12 4th harvest + virus
H7-H12 5th harvest + virus
  • Standard Curve:

090210 BCA Assay (Sunny) Standard Curve.png

  • Calculated Concentrations:
Sample Calculated Concentration (µg/mL)
0 µg/mL -116.3
25 µg/mL -130.5
125 µg/mL 32.7
250 µg/mL 192.1
500 µg/mL 443.8
750 µg/mL 763.4
1000 µg/mL 930.1
1500 µg/mL 1468.2
2000 µg/mL 1829.9
2.5 hr (-) virus 306.5
2.5 hr (+) virus 406.9
2nd harvest + virus 1112.2
3rd harvest + virus 687.7
4th harvest + virus 981.7
5th harvest + virus 516.1

BSA Assay Results

  • Notes: Done up by Crystal
Tube Concentration of BSA (mg/mL) Vol BSA Vol H2O Vol Bradford Abs value 100X Abs Calc. Conc (mg/mL)
1 0 0 100 3 0 0 -0.00112435
2 0.1 10 90 3 0.101 10.1 0.152958855
3 0.15 15 85 3 0.109 10.9 0.165163465
4 0.2 20 80 3 0.117 11.7 0.177368076
5 0.25 25 75 3 0.128 12.8 0.194149415
6 0.3 30 70 3 0.232 23.2 0.352809349
7 0.35 35 65 3 0.232 23.2 0.352809349
8 0.4 40 60 3 0.304 30.4 0.462650841
9 2.5 hr (-) 10 90 3 0.128 12.8 0.194149415
10 2.5 hr (+) 10 90 3 0.178 17.8 0.270428229
11 2nd H (+) 10 90 3 0.209 20.9 0.317721094
12 3rd H (+) 10 90 3 0.258 25.8 0.392474332
13 4th H (+) 10 90 3 0.284 28.4 0.432139316
14 5th H (+) 10 90 3 0.319 31.9 0.485534486

BSA Standard Curve.png