"
Team:Brown/Notebook/July8
From 2010.igem.org
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:
- Diluted 1000-fold (1 µl in 1 mL) of RFP control (5 µl)
- Non-diluted RFP control: 1 mL (5 µl)
- Gary’s buffers, ligase and pGEM (15 µl)
- 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.
