User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/05/24

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Contents

Objective

Subclone BSA into pTXB1

  • Use NheI and SapI cloning sites. This will fuse the BSA to a C-terminal intein and chitin binding domain (CBD).
  • Add C-terminal His8-tag after CBD.

Special Materials

BSA clone

  • BSA cDNA in pCMV-SPORT6
  • IMAGE clone ID # 8838824
  • E. coli DH10B TonA
  • glycerol stock frozen at -80°C

PCR primers

  • All primers from IDT
    • 100 nmol scale
    • HPLC purified
  • BSA cloning
    • BSA-NheI-5'
5'- CCC ACA AAA CTA TGG CTA GCA AGT GGG TGA CT-3'
Tm = 64.4°C
MW = 9842.4 g/mol
19.3 nmol, 0.19 mg
  • BSA-SapI-3'
5'-TGG TTG GCT CTT CTA CGG GCT AAG GCT GT-3'
Tm = 66.0°C
MW = 8946.8 g/mol
16.9 nmol, 0.15 mg
  • Kathryn Muratore 14:09, 11 August 2011 (EDT):This is the wrong sequence. It leaves an overhang of GCA at the 3' end, but the overhang for pTXB1 is TGC. I inverted these three bases when I designed the primer, and this explains why the ligation did not work.
  • His-tag cloning
    • ins_His_after_CBD
5'-CCT TGT GGC AGC TTC AAC ACC ACC ATC ATC ATC ACC ACC ACT GAC TGC AGG AAG GG-3'
Tm = 72.1°C
MW = 17083.1 g/mol
8.0 nmol, 0.14 mg
  • Rins_His_after_CBD
5'-CCC TTC CTG CAG TCA GTG GTG GTG ATG ATG ATG GTG GTG TTG AAG CTG CCA CAA GG-3'
Tm = 72.1°C
MW = 17398.3 g/mol
7.4 nmol, 0.13 mg

pTXB1

  • From NEB
  • 200 μg/mL

Experimental Plan

  1. Miniprep BSA clone
    1. streak clone on LB+Amp100 plates - today
      1. O/N @ 37°C
    2. inoculate 5 mL LB+Amp100 - tomorrow
      1. O/N @ 37°C
    3. Miniprep - Thurs
  2. Dilute primers to 100 μM with sterile H2O
    1. Dilute BSA cloning primers to 4 μM for subcloning PCR
    2. Dilute His-tag primers to 12.5 ng/μL for QuikChange
  3. BSA PCR - Thurs afternoon
    • Tanneal = 60°C
  4. analytical minigel - Fri afternoon
  5. purify by spin column - Fri afternoon
  6. QuikChange of pTXB1 (when plasmid arrives)
  7. DpnI digestion
    1. add 1μL DpnI to PCR tube
    2. 1h @ 37°C
  8. analytical minigel of pTXB1 PCR
  9. transform into DH10B
    • May need to make competent cells
  10. Miniprep
  11. sequence
  12. Sequential double-digest
    1. NheI digest
    2. SapI digest
    3. Phosphatase vector
    4. gel purify
  13. ligate
  14. transform DH10B
  15. miniprep
  16. sequence
  17. express

Today's benchwork

  1. Streak out BSA clone on LBAmp100
    • Plates made by TF and KW yesterday
→ 37C O/N


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