Team:TU Munich/Notebook/Labjournal

From 2012.igem.org

(Difference between revisions)
(→Saturday, 23th June)
(→Friday, 22th June)
Line 1: Line 1:
 +
==<center><p style="color:white; background-color:#0000EE"> '''Monday, 18th June''' </p></center>==
 +
 +
===<span style="color:#0000EE"> XXXX===
 +
 +
'''Investigator: Daniela, Saskia'''
 +
 +
==<center><p style="color:white; background-color:#0000EE"> '''Tueday, 19th June''' </p></center>==
 +
 +
===<span style="color:#0000EE"> XXXX===
 +
 +
'''Investigator: Daniela, Saskia'''
 +
 +
==<center><p style="color:white; background-color:#0000EE"> '''Wednesday, 20th June''' </p></center>==
 +
 +
===<span style="color:#0000EE"> XXXX===
 +
 +
'''Investigator: Daniela, Saskia'''
 +
 +
==<center><p style="color:white; background-color:#0000EE"> '''Thursday, 21th June''' </p></center>==
 +
 +
===<span style="color:#0000EE"> XXX===
 +
 +
'''Investigator: Daniela, Saskia'''
 +
==<center><p style="color:white; background-color:#0000EE"> '''Friday, 22th June''' </p></center>==
==<center><p style="color:white; background-color:#0000EE"> '''Friday, 22th June''' </p></center>==

Revision as of 17:35, 23 June 2012

Contents

Monday, 18th June

XXXX

Investigator: Daniela, Saskia

Tueday, 19th June

XXXX

Investigator: Daniela, Saskia

Wednesday, 20th June

XXXX

Investigator: Daniela, Saskia

Thursday, 21th June

XXX

Investigator: Daniela, Saskia

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 1.1 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
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Transformation with E.coli Xl1-Blue

Operation Sequence

  • melting of 100 µl Ca-competent E.coli XL1-Blue cells
  • addition of 1 µl of the Plasmid P7 pYes2_RFC25 MCS 1.2
  • 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

Retrieved from "http://2012.igem.org/Team:TU_Munich/Notebook/Labjournal"