#2
TRANSFORMATION OF E.COLI BACTERIA WITH PLASMID DNA

Use Top10 competent cells for small plasmids (3-10kb)
Use XL10gold competent cells  for large plasmids (>10kb) : pAdeasy or
large viral vectors, use XL10gold.
For plasmids of 30kb, may need electroporation competent bacteria (BJ5183)

DAY 1:
1. Thaw Top10 cells on ice, one vial (made in our lab, 130-150ul cells per vial
2. Pipet 1-5ul DNA onto cells, flick by hand. No pipetting up and down!
(use 1ul plasmid or 10ul ligation mix)
3. Incubate cell+DNA mix on ice for 30min. (25min ok, >30min not ok,
cells lose competency!)
Meanwhile, set up heat bath to 42C for heat shock step
4. "Heat Shock" step: Place tube at 42C for 45sec
5. Place tube now on ice for 2min
6. Add 900ul LB growth media > mix, transfer to a 15ml round bottom
snap cap plastic tube
7. Shake at 180-200rpm at 37C in bacterial incubator for 1 hour (45min ok)
8. Transfer the ~1ml LB/cell mix to a microfuge tube (pour ok)
9. Spin briefly 3min, 2500rpm > check that there is a pellet of bacterial cells
10. Remove ~700ul of the media from the top without disrupting pellet
11. Now ressuspend the ~300ul media left with the pellet
12. Place the cells on a LB-antibiotic plate and grow UPSIDE DOWN in
37C mini incubator

Check your plasmid vector to see if plates used have to be
LB-Ampicillin or LB-Kanamycin
For most plasmids > LB-Amp plates ok
For pShuttle plasmids, pDrive  > LB-kan plates are needed

DAY 2:
1. The next day: Take plates out of 37C incubator early (before 11am
for example) to avoid overgrowth and put upside down in the 4C fridge.
You should see selection of only  colonies that had the plasmid
containing antibiotic resistance gene.
2. ~4-6pm: we "pick" colonies using a sterile yellow pipette tip and
inoculate 5ml LB tubes
3.  Inoculated cultures grow overnight, shaking 37C ~180-200rpm

DAY3: The following day: We isolate DNA plasmid from each sample and
test to see if they contain our plasmid of interest.