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

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Pre-Lab Calculations

 * Done as listed in this [[Media:090203_Manipulation_of_Restriction_Digest.xlsx|spreadsheet]].
 * Volumes to be used (Table 1):
 * Calculations (Table 2):

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.
 * Refer to Pre-Lab Calculations (Table 1) for values used.

Part 5: Baculovirus Infection

 * Followed protocol on pg 55 to 58.
 * Schedule for harvesting:


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