Team:TU Munich/Notebook/Labjournal

From 2012.igem.org

(Difference between revisions)
(→2 Miniprep of E. coli XL1-Blue with pYes2 1.2 (continuation of 4))
(→2 Miniprep of E. coli XL1-Blue with pYes2 1.2)
Line 181: Line 181:
  * 2,5 µl pYes2 1.2/p13
  * 2,5 µl pYes2 1.2/p13
  * 37°C, 1 h.
  * 37°C, 1 h.
 +
 +
===<span style="color:#0000EE"> 2 Quick Change mutagenis to remove SpeI from pYES2 </span>===
 +
 +
'''Investigator: Ingmar, Volker'''
 +
 +
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
 +
 +
'''PCR'''<br>
 +
'''Reaction batch'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|4 µl
 +
|Plasmid P7 pYes2_RFC25 MCS 1.2 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'''
 +
{|cellspacing="0" border="1"
 +
|'''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

Revision as of 20:39, 25 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

1 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

2 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

1 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. Incubation overnight at 37°C 180 rpm.
  • Using a Quiagen kit a miniprep of the overnight culture was done.

2 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 of E. coli pYes2 1.2 was picked an transferred to 5 ml LB Amp. Incubation overnight at 37°C 180 rpm.

3 Transformation of 2 Biobricks into E. coli XL1-Blue

Investigator: Jeffery

Aim of the experiment: Transformation of Biobricks into E. coli for plasmid propagation for PCR with new RFC pre- and suffix primer in order to do protein fusions.

  • 2 Biobricks from the distribution kit were used: First, LexA (BBa_K105005, Plate 3 Well 9E) in the pSB1A2 plasmid with ampicillin resistance and second, the heme oxygenase (BBa_I15008, Plate 2 Well 13J) in the pSB2K3 plasmid with kanamycin resistance.
  • 10 µL of autoclaved H2O were added to each well on the distribution kit. The well immediately turned red which means that one does it right.
  • The now resuspended DNA liquids were transferred into a new ERG on ice.
  • CaCL2 competent E. coli XL1-Blue cells from the stock were gently defrezed on ice.
  • For each Biobrick 100 µL cells were used and pooled together with 2 µL of plasmid DNA in a ERG on ice.
  • Incubation on ice for 30 min.
  • 5 min heat shock at 37 °C.
  • Each ERG now is transferred in a new ERG prefilled with 1 mL of LB-medium and incubated in a cell-culture shaker at 37 °C for 45 min.
  • 100 µL of these cell suspension were plated on antibiotic selection plates (Ampicillin for LexA and Kanamycin for heme oxygenase).
  • The rest of the cell suspension is centrifuged at 13000 rpm for 60 s and the supernatant is discarded.
  • The pellet is resuspended with 100 µL for each ERG and is plated on another antibiotic selection plate
  • These 4 plates were put at 37 °C overnight

Monday, 25th June

2 Miniprep of E. coli XL1-Blue with pYes2 1.2

Investigator: Alois, Martin

Aim of the experiment: proof of successful removal of NgoMIV in the backbone of pYes2

Operation Sequence:

  • Mini prep of pYes2 1.2
  • Control digest of pYes2 1.2 and p13 (+ analytical gel electrophoresis: 90 V, 1 h: 
*  15 µl ddH20
*   2 µl NEBuffer4
* 0,5 µl NgoMIV
* 2,5 µl pYes2 1.2/p13
* 37°C, 1 h.

2 Quick Change mutagenis to remove SpeI 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 1.2 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

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