Team:TU Munich/Notebook/Labjournal

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===<span style="color:#0000EE"> Quick Change mutagenis to remove NgoMIV from pYES2 </span>===
===<span style="color:#0000EE"> Quick Change mutagenis to remove NgoMIV from pYES2 </span>===
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'''Investigator: Ingmar, Volker'''
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Aim of the experiment: Removal of a NgoMIV restriction site in the backbone of pYes2
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Operational sequence:
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* A single clone was picked an transferred to 5 ml LB Amp. Incubation overnight at 37°C 180 rpm.
==<center><p style="color:white; background-color:#0000EE"> '''Monday, 25th June''' </p></center>==
==<center><p style="color:white; background-color:#0000EE"> '''Monday, 25th June''' </p></center>==
===<span style="color:#0000EE"> Quick Change mutagenis to remove NgoMIV from pYES2 </span>===
===<span style="color:#0000EE"> Quick Change mutagenis to remove NgoMIV from pYES2 </span>===

Revision as of 18:16, 24 June 2012

Contents

Monday, 18th June

XXXX

Investigator: Daniela, Saskia

Tuesday, 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 into 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

Sunday, 24th June

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

Investigator: Ingmar, Volker

Aim of the experiment: Plasmid purification

Operation Sequence:

  • A single clone of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS was picked an transferred to 5 ml LB Amp on saturday evening.
  • Using a Quiagen kit a miniprep of the overnight culture was done.

Quick Change mutagenis to remove NgoMIV from pYES2

Investigator: Ingmar, Volker

Aim of the experiment: Removal of a NgoMIV restriction site in the backbone of pYes2

Operational sequence:

  • A single clone was picked an transferred to 5 ml LB Amp. Incubation overnight at 37°C 180 rpm.

Monday, 25th June

=== Quick Change mutagenis to remove NgoMIV from pYES2 ===

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