Meeting with Prem and Veeral-7
| Date | |
|---|---|
| Department | Wet Lab |
| Description | Discussed cyanobacteria protocols for receiving strains, reviving, growing |
| HP sub-branch | |
| Links/media | |
| Participants | |
| Property | |
| Property 1 | |
| Property 2 | |
| Type | Meetings |
Order plasmids from AddGene.
Revival Protocol:
→ Receive plates/slants containing solid medium.
→ Take one loop of the matted S. elongatus and place in liquid medium (preferred 10ml in 50ml flask; or 20ml in 100ml flask if done from a glycerol stock) and one loop for streaking in a new plate. BOTH MEDIA SHOULD CONTAIN APPROPRIATE SELECTIVE ANTIBIOTICS.(100mg/ml for Amp and Spec , 50 for Kan)
→ Grow for a few (3-4) days. Hopefully we see cultures. (We can store Michelle's plates at 4 degrees parallelly). If we don't get growth/we see a whitish colour we need to try again.
→ If the plates revive first, inoculate the medium with that culture. If the liquid revives, add 80ml of liquid and make it up to a 100ml culture. (OR: for backup, transfer 10ml to a 50ml flask and add 40ml of medium, preserving 10 ml to be kept under a light in the room sans shaking).
→At OD 0.6 - 0,8 under 730 nm, we are ready to prepare stocks. Autoclave a 25% glycerol solution as a base for the stocks.
Making Stocks:
→If a 100ml flask has an OD of 0.6, we treat a pellet derived by centrifugation (4000 RPM for 5-7 minutes) as having a total OD of 60 (0.6*100). Mix this pellet in a 12ml stock base (mix by tapping until it's homogenous). This gives us 5OD/ml (as 60/12 = 5). If instead it were 80, we'd have to use 16 ml of stock base.
→Each ml can be separated as an individual stock (store under -80)
→Revive one stock in 20 ml AFTER ONE DAY. If it doesn't revive, repeat process with your backup.
Making Plates;
→ To avoid using your stocks, centrifuge 1ml of your single revived stock as described above and streak the pellet on a plate to get single colonies. Grow until the colonies are visible and place at 4 degrees. Every 20-22 days, revive a colony in liquid and restreak. This plate replaces its predecessor.
PCR Verfication:
→Patch a plate from a single colony. Repeat for a few colonies.
→ Let the patch grow under the light. When it's grown quite a bit, take a loop and revive as above to get a green culture. Once the OD is 0.6-0.8, take 50 or 200 microlitres and heat at 95 degrees in an eppendorf for an hour.
→ Once that's cooled down, use one microlitre of this liquid for PCR verification of the presence of gene.
ALTERNATIVE:
→ Take 50 microlitres of water and add a loop from the patch. If the culture in the vial is light green, heat it and take a microlitre for PCR.
MAKE STOCKS OF PCR-CONFIRMED COLONIES.
gRNA cloning
→ Grow E. coli as delivered by AddGene. Make stocks. Take 30ml of LB media.