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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of ProtA⋅His to pEX 1.1.1 Digestion 1.1.2 Ligation 1.1.3 Transformation 1.2 PCR verification for Uppsala team

Andreas

Transfer of ProtA⋅His to pEX

Digestion

	Sample
10X FastDigest buffer	1
Plasmid DNA	7
dH2O	0
FD XbaI	1
FD PstI	1
	10 μl

• Incubation: 37 °C, 0:30
• Inactivation: 80 °C, 3:00

De-phosphorylated pre-digested/extracted pEX vector:

• 8 μl extracted and digested (X+P) pEX vector DNA
• 1 μl FD buffer
• 1 μl FastAP (alkaline phosphatase)

• Incubation: 37 °C, 0:30
• Inactivation: 80 °C, 3:00

Ligation

10X T4 Ligase buffer	2
Vector DNA	1
Insert DNA	11
dH2O	5
T4 DNA ligase	1
	20 μl

• Incubation: 22 °C, 1 h

Transformation

Including three more transformations

Modified quick-transformation protocol:

• 30 min on ice
• 50 μl BL21
  1. 2 μl pEX.ProtA⋅his ligation mix
  2. 0.5 μl pEX.IgGp
  3. 0.5 μl pEX.SOD.RyC
  4. 0.5 μl pEX.hS.RyC

PCR verification for Uppsala team

Helping the Uppsala team with a PCR verification of one of their assemblies.

Summed up their total construct length in pSB1x3 to 6566 bp.

• K1: C2 & C4 (x2)
• K2: C2 & C5 (x2)

Standard colony PCR settings:

• Elongation time: 10 min
• Annealing temp: 55 °C and 60 °C

PCR run ON.