User:Eric Ma/Notebook/MICB323 Lab Book/2009/02/03: Difference between revisions

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==Part 3: Measurement of DNA==
==Part 3: Measurement of DNA==
*Protocol followed as in lab manual pg. 47.
*Protocol followed as in lab manual pg. 47.
*A260: 0.227
*Calculations:
*20X A260: 4.54
**A260: 0.227
*[pDNA] (µg/mL): 227
**20X A260: 4.54
**[pDNA] (µg/mL): 4.54 x 50µg/mL = 227 µg/mL
 
==Part 4: Restriction Digest Manipulation==
*We need to get 0.2µg of DNA.
*Take (0.2µg)/(227µg/mL) = 0.88µL DNA.
 
 


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Revision as of 16:55, 3 February 2009

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Pre-Lab Calculations

  • Done as listed in this spreadsheet.
  • Volumes to be used:
Variable Tube # pDNA (µg) pDNA (µL) EcoR1 (µL) 10X React 3 (µL) 500 mM NaCl (µL) 500 mM Tris-HCl (µL) 100 mM MgCl2 (µL) dH2O (µL) Total Volume (µL)
[enzyme] 1 0.2 0.88 0.5 2 0 0 0 16.4 20
[enzyme] 2 0.2 0.88 1 2 0 0 0 15.9 20
[enzyme] 3 0.2 0.88 1.5 2 0 0 0 15.4 20
[enzyme] 4 0.2 0.88 2 2 0 0 0 14.9 20
[NaCl] 1 0.2 0.88 0.5 2 1 0 0 15.4 20
[NaCl] 2 0.2 0.88 0.5 2 2 0 0 14.4 20
[NaCl] 3 0.2 0.88 0.5 2 3 0 0 13.4 20
[NaCl] 4 0.2 0.88 0.5 2 4 0 0 12.4 20
Component Tube [NaCl] in 10X React 3 (mM) [NaCl] Provided (mM) [NaCl] Wanted (10X) (mM) Extra [NaCl] needed (mM) 500 mM NaCl
NaCl 1 500 500 1000 500 1
NaCl 2 500 500 1500 1000 2
NaCl 3 500 500 2000 1500 3
NaCl 4 500 500 2500 2000 4

Part 1: Plasmid Extraction

  • Electroporation transformed: No growth.
  • EM#1 - CaCl2 untransformed: Growth.
  • EM#2 - CaCl2 transformed: Growth.
  • I will use Sunny's culture's DNA.


  • Entire ON culture - spin @8000rpm @3min. Performed split up into two spins in same tubes.
    • Note: Found out that not all of the EM#2 was transferred into the 2nd spin. There will be 750µL less (i.e. 1/4 less) cells present. This may reduce [pDNA] obtained in the end; expected to be 1/4 less compared to EM#1. I'm not sure, but may be useful to measure later on.

Part 2: R.E. Digest

  • Protocol followed as in lab manual pg. 46.
  • No problems encountered.

Part 3: Measurement of DNA

  • Protocol followed as in lab manual pg. 47.
  • Calculations:
    • A260: 0.227
    • 20X A260: 4.54
    • [pDNA] (µg/mL): 4.54 x 50µg/mL = 227 µg/mL

Part 4: Restriction Digest Manipulation

  • We need to get 0.2µg of DNA.
  • Take (0.2µg)/(227µg/mL) = 0.88µL DNA.



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