User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/18

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Objective

Spin down and freeze cells from last night

Test QuikChange with pTXB1 and more cycles

Bench work

  1. Spun down 2 x 500mL of both expressed MBP w/ and w/o intein (pMXB10 and pMAL-pIII) at 3810 rpm for 10 minutes at 4°C
    • Poured off and discarded supernatant and inverted to dry
    • Resuspended pMXB10 pellets in total of 25mL of column buffer 20mM TrisCl + 500mM NaCl pH 8.5
    • Resuspended pMAL-pIII pellets in total of 100mL of resuspension buffer 30mM TrisCl + 20% w/v sucrose pH 8.0
    • Quick froze pMXB10 tube (~30mL) with liquid nitrogen and stored at -80°C
    • Quick froze two tubes of pMAL-pIII with liquid nitrogen/stored at -80°C, and placed two other tubes in -20°C freezer
  2. Test 2-stage QuikChange with pTXB1 10ng/μL dilution from 6/7/11:
    • 10 cycle first stage (up from 5 before and ten times the single cycle in original protocol) and 18 cycle second stage
    • 7 minute extension step

Results

  • The MBP-bearing cells were grown in the absence of glucose, which is needed to suppress the expression of, among others, amylase. Amylase will breakdown the amylose resin, leading to poor purification. The growth and expression of the host cells will have to be repeated with 0.2% glucose in the growth media
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