User:Mark X. Ling/Notebook/Micb 323/2009/02/03

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Lab 4: Quick Plasmid Isolation, restriction digest and Insect tissue culture.

Part 1: Plasmid Isolation

  • cells were isolated from: Lab 3 part 1, white and blue colony, and part 2A colony(plate 2A3)

Part2:Restriction Digest of plasmid

  • no change same as in lab manual.

Section 1: Normal restriction digest

  • time incubated: 2:09
  • temperature incubated:37 oC
 DNA content:
 **measurement done on Part 1 white colony. ligated.
 *A260nm:0.200
 **more than 0.15 so may not be accurate.
 *A280nm:0.107
 *A260/A280:1.89
 *concentration DNA: 2000ug DNA/mL


Tube Volume plated final plated dilution Blue colonies White colonies Total colonies % Transformed White/Blue Ratio
1 200 uL 0.2 59 42 101 0.415841584 0.711864407
2 100 uL 0.1 17 16 33 0.484848485 0.941176471
3 50 uL 0.05 3 3 6(ESPC) 0.5 1
Average 0.450345035 0.826520439

Section 2: Modified Restriction digest

EcoR1:

Tube # Plasmid (ug) Plasmid (uL) EcoR1 (uL) 10x React (uL) 500mM NaCL (uL) 500mM tris-HCl (uL) 100mM MgCl2 (uL) dH2O (uL)
1 0.2 2 0.5 2 0 0 0 15.5
2 0.2 2 0.2 2 0 0 0 15.8
3 0.2 2 2 2 0 0 0 14
4 0.2 2 5 2 0 0 0 11

NaCl:

Tube # Plasmid (ug) Plasmid (uL) EcoR1 (uL) 10x React (uL) 500mM NaCL (uL) 500mM tris-HCl (uL) 100mM MgCl2 (uL) dH2O (uL)
1 0.2 2 5 2 2 0 0 9
2 0.2 2 5 2 5 0 0 6
3 0.2 2 5 2 7 0 0 4
4 0.2 2 5 2 11 0 0 0
  • digests incubated at: 2:58
  • incubated at:37/38.2 oC
  • MISTAKE: used 4ug of DNA instead of 0.2ug

Part 3: Insect Tissue culture infected with Baculovirus

  • Time incubated on room temperature:325
  • room temperature:22 oC
  • harvest schedule=> Tue:Eric => Wed:Mark => Thu:Crystal => Fri: Mark => Mon:Sunny

Post Lab comments

  • Miscalculation DNA was done.
    • had too much DNA in the RE enzyme modification
    • all samples are of extreme low. Will not be useful in assessment of the need of each components.
  • DNA isolation went smoothly
    • seemed to have good amount BUT the A260nm was higher than 0.200, so there may be RNA contamination.