General cloning strategy
Make sure you have some cells on a fresh plate that you’re going to transform
into, so you can grow up o/n culture and then make competent.
1. Get insert:
- PCR insert (setup plus 3 hours), run on gel (1.5 hours), geneclean (1
hour), elute one band in 50ul (end up with 40 ul)
2. Cut
-
insert and vector
Take all 40 odd ul insert and digest in final vol of 50ul
Digest 2ul vector DNA in 20ul, 37oC for 2 hours
Now heat inactivate reaction at 80oC for 20mins, cool down, add 1ul CIP to
20ul vector reaction (not entirely necessary if cut with 2 enzymes), 37oC
for 1 hour, run on gel (1.5 hours), geneclean vector fragment (1 hour)
into 50ul AND INSERT reaction into 30ul (no need to run this on gel ‘cos
won’t be able to discriminate between uncut insert anyway)
3. Ligate
- take all cut insert (30ul) and ligate with (1/4 or 1/3 vector) e.g. 8ul of the
cut vector, in 50ul total, 15oC overnight or 15mins if quickstick
- grow up o/n culture for making competent cells next day
4. Transform
- Either spend morning making competent cells from o/n culture OR
1. defrost on ice, about 20mins
2. add up to 20ul DNA (ideally 10pg):
a. high copy plasmids – 1ul
b. low copy – 2-5ul
c. ligation mixes – 20 to 50ul of 50ul ligation mix
3. keep on ice for 1 hour
4. heat shock as normal (2min@42oC, 2min on ice)
5. add 800ul fresh LB, no Ab
6. incubate at 37oC, 1 hour to allow Ab res. genes to express
7. Spread plate (30 mins) 100
plate and incubate at 37 oC o/n. Spin down remaining volume and resuspend in ~100ul – then spread plate on a separate plate containing
antibiotics.
-
if this fails twice, try adding 2% glucose to reduce expression levels (if
product is toxic)