Cloning + Mutagenesis

PCR insert
10X Buffer 10
5M AC rich? 5
F primer (10uM) 1.25
R primer 1.25
dNTP 1
template 2
Taq polymerase 1
water 28.5
50ul
95C 5min
95C 1min
58C 1min
72C 1.5min
(repeat from 2 34x)
72C 7min
4C forever

Gel purifcation, elute in 38ul

Restriction enzyme digest
10X Buffer 5
10X BSA 5
enzyme 1
enzyme 1
plasmid 38
50ul
37C for 2h

PCR purification

Ligation
3 insert: 1 vector

Transformation
1. Add 5-15ul plasmid to 100ul competent cells (DH5a). Mix by flicking tube
2. Incubate on ice for 30min
3. Heat shock bacteria in 42C for exactly 30s, then put on ice
4. Add 250ul of pre-warmed(RT) SOC
5. Lie tube sidways and shake for 1h
6. Reduce to 100ul and plate

PCR check (colony PCR)
10X Buffer 2
MgCl2 1.2
F primer 0.5
R primer 0.5
Taq 0.2
dNTP 0.4
water 15.2
20ul
95C 5min
95C 1min
55C 1min
72C 1.5min
(repeat from 2 34x)
72C 7min
4C forever

Culture 3ml of bacteria
Miniprep

Restriction enzyme digest
10X Buffer 2
10X BSA 2
enzyme 0.2
enzyme 0.2
plasmid 5
water 10.6
20ul

Sequencing
3.2ng primers/ 200-300ng DNA in 6ul
primers (diluted 5 times) 1.6
plasmid 4.4
6.0ul

Mutagenesis
PCR
10x buffer 5
plasmid (diluted 10x) 2 (10ng/ul)
F primer 1.25
R primer 1.25
dNTP 1
Polymerase (Pfu) 1
water 38.5
50ul
95C 30s
95C 30s
55C 1min
68C 8min (1min/1kb of plasmid)
(repeat from 2 17x)

Digestion
Dpn1 1ul
PCR product 50ul

Mass culture
3 tubes of 20ml of LB/amp each
Miniprep 5ml/ column
Combine all elutions
Final conc ~300ng/ul