"

From 2010.igem.org

• Home
• Projects
  • Light-Pattern Control
  • E. Cargo (Tat-PTD)
  • BioBricks
  • Protocols
  • Notebook
  • Obstacles/Lessons
• Modeling
  • ODEs
  • Parameters
  • Results
  • Light induction device
• Human Practices
  • Survey
  • Community Outreach
  • Journal Club
  • Safety
• Team

Thursday, July 8 2010

Transformation of pPTPi into BL-21s

Note: XL1 Blue is endogenously resistant to Tetracycline and cannot be used for selection of a Tet-R plasmid.

• 150 µl BL-21 (stored at -80°C) in each tube (Z)
• Added 1 µl RFP control Plasmid
• 2.5 µl pPTPi

Incubated on ice for 24 minutes.

Heat shock at 42°C for 2 minutes.

Added 0.5 mL LB to each tube and placed in incubator at 3:00 PM.

4:00 PM – Spun at 3500 RPM for 4.5 minutes.

Resuspended in 100 µl LB per tube.

Plated:

• RFP Control -- 50 µl on tet+ plate, 50 µl on amp+ plate
• pPTPi -- 50 µl on tet+ plate, 50 µl on tet+ plate

Placed at 37°C at 4:27 PM. Overnight.

Cell Transformation of LovTAP into BL-21 and XL-1

• Spread 40 µl of 0.1 M IPTG over five Kanamycin plates and one Ampicillin plate.
• Thawed three centrifuge tubes with 150 µl competent XL1 Blues and three tubes with 150 µl competent BL-21s.
• Added 1 µl control RFP to 1 tube of XL1B and 1 tube BL-21s.
• Added 2 µl LovTAP (rehydrated from well 20-F, plate 3 of distribution with 10 µl dH2O) to two tubes of XL1-Blues and two tubes of BL-21s.
• Incubated tubes for 22 minutes on ice
• Heat shock for 2 minutes at 42°C
• Removed from bath and placed on ice, added 500 µl LB media to each tube and placed in incubator.
• Removed from incubator, pelleted cells in centrifuge and resuspended in 100 µl fresh LB per tube.
• Plated cells on corresponding plates

Left plates on bench (room temperature) over weekend.

Redo Ligation (WillRS + pGEM T Easy)

• 7.5 µl 2X DNA ligase buffer (rapid)
• 1 µl T4 DNA ligase
• 0.5 µl vector (pGEM T Easy)
• 1.5 µl insert (WillRS)
• 4.5 µl MilliQ H2O

15 µl total volume

Ran parallel ligation reactions, using Gary’s and our reagents (Ligase, buffer, and pGEM) – 6:30 PM.

1’ RT.

Transformation

7:40 PM Did four transformation tubes:

1. Diluted 1000-fold (1 µl in 1 mL) of RFP control (5 µl)
2. Non-diluted RFP control: 1 mL (5 µl)
3. Gary’s buffers, ligase and pGEM (15 µl)
4. iGEM’s buffers, ligase, and pGEM (15 µl)

Used Adrian’s transformation protocol:

• Thaw competent cells on ice – each tube with 150 µl XL1-B
• Adjust final volume of cells to 100 µl with cold 50 mM CaCl2
• Add ligation mixture (if in low melt, heat to 67°C, and then transfer to 37°C)
• Incubate on ice for 30 minutes.
• Heat shock at 42°C for 1 minute.
• Incubate 2” on ice.
• Add 100 µl LB

'Growing in a shaking incubator for an hour is only for baterocidal antibiotics; amp is bacteriostatic, so don’t do it for Amp.'

Blue-White Screen

• Add 50 µl X-gal (20 mg/mL stock) to cells
• Add 2 µl IPTG (1M stock) to cells
• Plate out on four amp+ plates
• Grow overnight at 37°C

To further develop color after colonies grow up, allow growth at 4°C for a few more hours. There is a chance that the fragment of interest does not introduce a STOP codon, resulting in lighter blue colonies. If unsure, or there are comparable numbers of both colonies, test a few of both.