Melbourne/Lab Notebook gv F1
From 2007.igem.org
Testing Floatation
- Growing up cells from glycerol stocks overnight in 5mL LB amp
- Prepare 250ml conical shaker flasks by cleaning and autoclave.
- Add 50mL of LB Amp to each of two flasks.
- Add 1M IPTG to one flask to make final concentration of 1mM IPTG, Lable with I.
- Transfer 1mL of overnight culture to each flask.
- Grow overnight at 37degC on a shaker.
- Next morning transfer to 50ml falcon tubes.
- Spin down the cells gently 25minutes at 1000rpm in Eppendorph 5810R centrifuge.
- Poor off and discard the LB, maintaining the pellet and a residual ammount of LB 1ml about.
- Replace LB with 50ml of 10g/L NaCl filtered through a 0.22um filter.
- Resuspend the cells by shaking.
- Leave at room temperature and observe the settling over the next week.
Example pNL29(orininal plasmid) and Tube(64) the IPTG promoted version of BBa_I750016
In the following series of photos the tubes are in pairs. The outer pairs are uninduced (no IPTG), the inner pairs are induced. The tubes left of centre are the original pNL29 plasmid in DH5a cells, the tubes on the right are 41C plasmids (promoted BBa_I750016) in XL10 gold. We believe the XL10-gold is permanenently inducing the expression even without IPTG, this seems to be confirmed by the latter experiment where the plasmid was retransformed into DH5alpha cell.
