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

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Pre-Lab Calculations Done as listed in this spreadsheet. Volumes to be used (Table 1): 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 Calculations (Table 2): 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. Refer to Pre-Lab Calculations (Table 1) for values used. Part 5: Baculovirus Infection Followed protocol on pg 55 to 58. Schedule for harvesting: Day: Person Tuesday Eric Wednesday Mark* Thursday Crystal Friday Mark Monday Sunny