"

From 2012.igem.org

Contents 1 Friday, 22th June 1.1 Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS 2 Saturday, 23th June 2.1 Quick Change mutagenis to remove NgoMIV from pYES2 3 PCR reaction batch 3.1 PCR cycling parameters

Friday, 22th June

Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS

Investigator: Ingmar, Volker

Aim of the experiment: Plasmid amplification

Operation Sequence:

• melting of 100 µl Ca-competent E.coli XL1-Blue cells
• addition of 1 µl of the Plasmid pKS2µHyg-PAL-4Cl-CHS
• incubation for 30 min on ice
• heat shock for 5 min at 37 °C
• transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
• plate 100 µl on an Amp-LB-plate
• sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Saturday, 23th June

Quick Change mutagenis to remove NgoMIV from pYES2

Investigator: Ingmar, Volker Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR reaction batch

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P7 pYes2_RFC25 MCS template
0.5 µl	1:10 dilution of O38 (10 pmol/µL)
0.5 µl	1:10 dilution of O39 ((10 pmol/µL)
17 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		68°C	6 min