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

Southern Hybridization

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
A0.1040.10.1020.0980.0970.1060.8390.8410.8140.8920.8810.918
B0.1050.1050.1010.10.1010.1092.8942.8132.8192.8120.0350.034
C0.1730.1670.1640.1630.1590.1612.8352.7882.8472.8010.0340.034
D0.2270.230.2170.2090.2080.2152.7462.7462.7852.7470.0340.035
E0.3590.3430.3360.330.3190.3262.732.7862.7282.6880.0340.034
F0.4570.4460.4920.4530.4370.4262.6772.6672.6752.6690.0350.033
G0.5440.5390.5330.5420.5390.5292.7582.7342.7292.7140.0340.034
H0.7030.6860.7230.7540.7540.7780.0340.0340.0340.0350.0340.034
  • Guide to plate:
Wells Sample
A1-A60 µg/mL
B1-B625 µg/mL
C1-C6125 µg/mL
D1-D6250 µg/mL
E1-E6500 µg/mL
F1-F6750 µg/mL
G1-G61000 µg/mL
H1-H61500 µg/mL
A7-A122000 µg/mL
B7-B122.5 hr (-) virus
C7-C122.5 hr (+) virus
D7-D122nd harvest + virus
E7-E123rd harvest + virus
F7-F124th harvest + virus
G7-G125th harvest + virus
H7-H12N/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/mL111.2
250 µg/mL246.1
500 µg/mL545.2
750 µg/mL840.4
1000 µg/mL1058.3
1500 µg/mL1554.1
2000 µg/mL1887.0
2.5 hr (-) virus6887.8
2.5 hr (+) virus6845.3
2nd harvest + virus6688.6
3rd harvest + virus6630.2
4th harvest + virus6475.4
5th harvest + virus6632.1

Sunny's Plate (Wild-Type Virus)

Row\\Column 1 2 3 4 5 6 7 8 9 10 11 12
A0.1010.1110.0980.0990.1050.10.7370.7950.7910.7310.790.752
B0.1010.0970.10.0910.0980.0980.0340.0340.0340.0340.0350.034
C0.1580.1530.1520.1540.1440.1580.3350.2360.2040.2110.040.034
D0.2120.210.2090.2050.1970.2120.3840.2570.2310.2510.0340.034
E0.3340.2990.2870.2870.2870.2660.6240.4460.3650.650.0340.034
F0.4090.420.4040.3950.3980.3880.5160.3310.2920.3670.0330.033
G0.4690.4880.410.4290.4760.4830.5770.720.3130.2970.0340.034
H0.6840.7080.6220.5910.6090.6420.4150.3250.2650.2670.0340.034
  • Guide to plate:
Wells Sample
A1-A60 µg/mL
B1-B625 µg/mL
C1-C6125 µg/mL
D1-D6250 µg/mL
E1-E6500 µg/mL
F1-F6750 µg/mL
G1-G61000 µg/mL
H1-H61500 µg/mL
A7-A122000 µg/mL
B7-B12N/A
C7-C122.5 hr (-) virus
D7-D122.5 hr (+) virus
E7-E122nd harvest + virus
F7-F123rd harvest + virus
G7-G124th harvest + virus
H7-H125th harvest + virus
  • Standard Curve:

  • Calculated Concentrations:
Sample Calculated Concentration (µg/mL)
0 µg/mL-116.3
25 µg/mL-130.5
125 µg/mL32.7
250 µg/mL192.1
500 µg/mL443.8
750 µg/mL763.4
1000 µg/mL930.1
1500 µg/mL1468.2
2000 µg/mL1829.9
2.5 hr (-) virus306.5
2.5 hr (+) virus406.9
2nd harvest + virus1112.2
3rd harvest + virus687.7
4th harvest + virus981.7
5th harvest + virus516.1

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)
100100300-0.00112435
20.1109030.10110.10.152958855
30.15158530.10910.90.165163465
40.2208030.11711.70.177368076
50.25257530.12812.80.194149415
60.3307030.23223.20.352809349
70.35356530.23223.20.352809349
80.4406030.30430.40.462650841
92.5 hr (-)109030.12812.80.194149415
102.5 hr (+)109030.17817.80.270428229
112nd H (+)109030.20920.90.317721094
123rd H (+)109030.25825.80.392474332
134th H (+)109030.28428.40.432139316
145th H (+)109030.31931.90.485534486


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