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 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
- 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
- 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.
- 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
- 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
Experimental Plan
- Miniprep BSA clone
- streak clone on LB+Amp100 plates - today
- O/N @ 37°C
- inoculate 5 mL LB+Amp100 - tomorrow
- O/N @ 37°C
- Miniprep - Thurs
- Dilute primers to 100 μM with sterile H2O
- Dilute BSA cloning primers to 4 μM for subcloning PCR
- Dilute His-tag primers to 12.5 ng/μL for QuikChange
- BSA PCR - Thurs afternoon
- analytical minigel - Fri afternoon
- purify by spin column - Fri afternoon
- QuikChange of pTXB1 (when plasmid arrives)
- DpnI digestion
- add 1μL DpnI to PCR tube
- 1h @ 37°C
- analytical minigel of pTXB1 PCR
- transform into DH10B
- May need to make competent cells
- Miniprep
- sequence
- Sequential double-digest
- NheI digest
- SapI digest
- Phosphatase vector
- gel purify
- ligate
- transform DH10B
- miniprep
- sequence
- express
Today's benchwork
- Streak out BSA clone on LBAmp100
- Plates made by TF and KW yesterday
- → 37C O/N
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