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From 2012.igem.org

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Contents 1 Monday, 18th June 1.1 XXXX 2 Tuesday, 19th June 2.1 XXXX 3 Wednesday, 20th June 3.1 XXXX 4 Thursday, 21th June 4.1 XXX 5 Friday, 22th June 5.1 Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS 6 Saturday, 23th June 6.1 Quick Change mutagenis to remove NgoMIV from pYES2 7 Sunday, 24th June 7.1 Miniprep of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS 7.2 Quick Change mutagenis to remove NgoMIV from pYES2 7.3 Transformation of 2 Biobricks into E. coli XL1-Blue 8 Monday, 25th June 8.1 Miniprep of E. coli XL1-Blue with pYes2_RFC25 MCS 1.2 8.2 Quick Change mutagenis to remove SpeI from pYES2_RFC25 MCS 1.2 8.3 Picking of E. coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase) 9 Tuesday, 26th June 9.1 Quick Change mutagenis to remove SpeI from pYes2_RFC25 MCS 9.2 PCR of PAL, 4CL, CHS, OMT (Coumaryl-CoA) 9.3 Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase) 10 Friday, 29th June 10.1 Preparative digest of PCR-products of 4CL, CHS and OMT 10.2 Preparative Gelelectrophoresis of PCR-products of 4CL, CHS 10.3 Quick Change mutagenesis to remove PstI in URA3 from pYES2_RFC25 MCS 1.2 11 Saturday, 30th June 11.1 Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS 12 Sunday, 1th July 12.1 Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS 12.2 Quick Change mutagenesis to remove PstI in the 2µ ori from pYES2_RFC25 MCS 13 Monday, 2th July 13.1 Repetition of PCR of PAL 13.2 PCR of LexA with primers including the RFC25 pre- and suffix 14 Tuesday, 3th July 14.1 Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix 14.2 LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase

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

• The vector resulting from the PCR-product was named pYes2_RFC25 MCS 1.2.
• 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. Incubation overnight at 37°C 180 rpm.
• 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 of E. coli pYes2_RFC25 MCS 1.2 was picked an transferred to 5 ml LB Amp. Incubation overnight at 37°C 180 rpm.

Transformation of 2 Biobricks into E. coli XL1-Blue

Investigator: Jeffery Truong

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

Miniprep of E. coli XL1-Blue with pYes2_RFC25 MCS 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. The resulting purified DNA is P33.
• Control digest of pYes2_RFC25 MCS 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.

Quick Change mutagenis to remove SpeI from pYES2_RFC25 MCS 1.2

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 P33 template
0.5 µl	1:10 dilution of O44 (10 pmol/µL)
0.5 µl	1:10 dilution of O45 ((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

• The procedure was furthermore applied to P13 and P14.
• The vector resulting from the PCR-product was named pYes2_RFC25 MCS 1.3.
• 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

• This operation sequence was applied to the PCR prducts of P33, P13 and P14 respectively.
• melting of 100 µl Ca-competent E.coli XL1-Blue cells
• addition of 1 µl of the PCR product
• 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

Picking of E. coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Investigator: Jeffery Truong

Aim of the experiment: Picking colonies from transformed E. coli XL1-Blue, 4x picked for each Biobrick.

• pSB1A2 plasmid with BBa_K105005 (LexA): Colonies were on both ampicillin selection plates, the one with diluted cell suspension and the one with concentrated E. coli cell suspension. Typical E. coli colony morphology. Picking was performed on the plate with diluted cell suspension.

• pSB2K3 plasmid BBa_I15008 (heme oxygenase): Colonies were only on the kanamycin selection plate with concentrated cell suspension. The one with diluted susepension was empty. Typical but very small E. coli colonies. Picking was performed from the first plate.

• Picked pipette tips was transferred into a special cell-culture tubes with air-permeable, but sterile cover. In each tube 4 mL of LB-medium + ampicillin (???)(for pSB1A2) or kanamycin (35 mg/mL) (for pSB2K3).

• 4 colonies for each Biobrick was picked; total: 8 tubes overnight culture.

• These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Tuesday, 26th June

Quick Change mutagenis to remove SpeI from pYes2_RFC25 MCS

Investigator: Ingmar

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

Operational sequence:

• For each transformation of the PCR-products of P14 and P33 a single clone was picked an transferred to 6 ml LB Amp. Incubation overday at 37°C 180 rpm. The transfomation with the PCR product of P13 was not successfull. Therfore no clone could be picked.
• Using a Quiagen kit a miniprep of the overnight culture was done.
• The resulting purified DNA was aliquoted in new tubes labeled as follows:

PCR product of P33(transformation done by Ingmar): P29

PCR product of P33(transformation done by Saskia&Jara): P30

PCR product of P14(transformation done by Ingmar): P31

PCR product of P14(transformation done by Saskia&Jara): P32

• Afterwards a control digestion of P29-P32 was done.

Reaction batch

Plasmid	P29	P30	P31	P32
NEB4 buffer	2 µl	2 µl	2 µl	2 µl
DNA	2,5 µl	2,5 µl	5 µl	5 µl
SpeI-HF	0.25 µl	0.25 µl	0.25 µl	0.25 µl
NgoMIV	0.25 µl	0.25 µl
ddH2O	15 µl	15 µl	12.75 µl	12.75 µl
Sum	20 µl	20 µl	20 µl	20 µl

• Incubation at 37 °C for 1h.
• Verification of control digest by agarose gel electrophoresis:

20 µl of each digest product was mixed with 2 µl DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V.

PCR of PAL, 4CL, CHS, OMT (Coumaryl-CoA)

Investigator: Daniela, Mary

Determination of concentration of plasmids (Nanodrop): c(pKS2µHyg-PAL-4CL-CHS) = 500 ng/µl c (pOMT) = 20 ng/µl

PCR:

Name of tube	Enzyme	consensus (+)/ consensless (-)	used Oligos
CHS -	CHS	-	O13 and O24
CHS +	CHS	+	O23 and O24
PAL -	PAL	-	O15 and O16
PAL +	PAL	+	O22 and O16
OMT -	OMT	-	O17 and O26
OMT +	OMT	+	O25 and O26
4CL -	4CL	-	O19 and O20
4CL +	4CL	+	O21 and O20

Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
4 µl	dNTP's (each 2.5 mM)
0.5 µl	Pfu Ultra II (2.5 U/µL)
5 µl	1:10 dilution of used forward primers (10µM)
5 µl	1:10 dilution of used reversed primers (10µM)
1 µl	DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL or pOMT 20 ng/µL)
29.5 µL	bidest. sterile Water

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	5 min (and adding Pfu Ultra after 3 min)
2	30	95°C	30 sec
		46°C	2.5 min
		72°C	1.5 min
3		72°C	5 min

PCR purification

• Purification was done using QIAquick PCR Purification Kit (250)

Analytical Gel Electrophoresis:

-> going on with CHS, 4CL and OMT; the PCR of PAL will be repeated

Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Investigator: Jeffery Truong, Georg Schützinger

Aim of the experiment: Plasmid isolation from the picked transformed overnight E. coli cells with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase).

• The LB-medium with antibiotics of every tube was opaque which means that the picked cells were successfully inoculated.

• Centrifugation step at 5000 rpm for 10 min at 16 °C.

• Every single step now was performed on ice.

• Miniprep (Qiagen Qiaprep spin) after manufacturer's protocoll.

• Analytical restriction master mix was prepared after following scheme (Using XbaI and PstI):
  • 4.4 µL XbaI
  • 4.4 µL PstI
  • 17.6 µL Tango-buffer (10x)
  • 132 µL ELGA H2O

• 17.5 µL from the master mix was poooled together with 2.5 µL of plasmid DNA. That corresponds to 2.5 µL of plasmid DNA, 0.25 µL XbaI, 0.25 µL PstI, 2 µL Tango-buffer (10x), 15 µL ELGA H2O.

• Incubation at 37 °C for 120 min on a ERG heating unit.

• BUT: error was performed during preparing the digested plasmid DNA for analytical gelelectrophoresis in the dilution step. 1:10 dilution of analyctical probe with DNA loading buffer:
  • 3.3 µL sample (contains already 1x loading buffer!)
  • 0.7 µL loading buffer (?x)
  • 6 µL TAE-buffer

• Should have done: 3 µL sample + 1 µL loading buffer (10x) + 6 µL TAE-buffer (1x)

• DNA-ladder preperation: 10 µL ladder stock solution + 10 µL DNA loading buffer + 80 µL TAE-buffer. 10 µL of this solution was pipetted in one gel pocket of the prepared 1% agarose gel including ehtiudiumbromid.

• 20 µL of each samples were also pipetted into the gel pockets.

• The gel pockets were pipetted after following scheme:

Heme oxygenase (colony 1)

Heme oxygenase (colony 2)

Heme oxygenase (colony 3)

Heme oxygenase (colony 4)

DNA-ladder

LexA (colony 1)

LexA (colony 2)

LexA (colony 3)

LexA (colony 4)

• Gel electrophoresis at 90 V

• After 20 min the resolution was still poor; 20 min longer.

• Analytical Gel okay, but samples interchanged

Friday, 29th June

Preparative digest of PCR-products of 4CL, CHS and OMT

Investigator: Katrin, Mary

each digestion will dure 2.5h at 37°C

• CHS: digestion with Xba1 and HF-Age1 (both NEB)

volume	reagent
25µl	PCR-product
5µl	Buffer NEB4
0.5µl	BSA
1µl	Xba1 (NEB; 20u/µl)
1µl	HF-Age1 (NEB; 20u/µl)
17.5µl	bidest. sterile H2O

• OMT: digestion with Xba1 and HF-Age1 (both NEB)

volume	reagent
25µl	PCR-product
5µl	Buffer NEB4
0.5µl	BSA
1µl	Xba1 (NEB; 20u/µl)
1µl	HF-Age1 (NEB; 20u/µl)
17.5µl	bidest. sterile H2O

• 4CL: digestion with Xba1 and Pst1 (both Fermentas)

volume	reagent
25µl	PCR-product
5µl	Buffer Tango
2µl	Xba1 (Fermentas; 10u/µl)
3µl	Pst1 (Fermentas; 10u/µl)
15µl	bidest. sterile H2O

Preparative Gelelectrophoresis of PCR-products of 4CL, CHS

Investigator: Katrin, Mary

picture follows!

Gelextraction of 4CL+, 4CL-, CHS+, CHS- (bands are as expected; +=with consensus-sequence, -=without consensus-sequence)

DNA-purification with Kit from Quiagen

Quick Change mutagenesis to remove PstI in URA3 from pYES2_RFC25 MCS 1.2

Investigator: Ingmar

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 P29 template
0.5 µl	1:10 dilution of O40 (10 pmol/µL)
0.5 µl	1:10 dilution of O41 ((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
		67°C	6.5 min

• The procedure was furthermore applied to P31.
• 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

• This operation sequence was applied to the PCR prducts of P29 and P31 respectively.
• melting of 100 µl Ca-competent E.coli XL1-Blue cells on ice
• addition of 1 µl of the PCR product
• 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, 30th June

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

Investigator: Ingmar

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

Operational sequence:

• For each transformation of the PCR-products of P29 and P30 a single clone was picked an transferred to 6 ml LB Amp. Incubation overnight at 37°C 180 rpm. The transfomation with the PCR product of P31, P32 and P33 was not successfull. Therfore no clone could be picked from these plates and four instead of one clone was picked from the plates containing the transformations of P29.

Sunday, 1th July

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

Investigator: Ingmar

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

Operational sequence:

• Using a Quiagen kit a miniprep of the overnight culture was done.
• The resulting purified DNA was aliquoted in new tubes labeled as follows:

1st PCR product of P29: P34

2nd PCR product of P29: P35

3rd PCR product of P29: P36

4th PCR product of P29: P37

PCR product of P30: P38

• Afterwards a control digestion of P34-P38 was done.

Reaction batch

Plasmid	P34	P35	P36	P37	P38
Fermentas 10x R buffer	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl
DNA	2 µl	2 µl	2 µl	2 µl	5 µl
PstI	0.25 µl	0.25 µl	0.25 µl	0.25 µl	0.25 µl
ddH2O	17.25 µl	17.25 µl	17.25 µl	17.25 µl	14.25 µl
Sum	20 µl	20 µl	20 µl	20 µl	20 µl

• Incubation at 37 °C for 1h.
• Verification of control digest by agarose gel electrophoresis:

20 µl of each digest product was mixed with 4 µl 6x DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V.

• All digest products show the expected two bonds at 3526 bp and at 2332 bp. The Miniprep product P35 was chosen to be used for the further Quickchange Mutagenesis.

Quick Change mutagenesis to remove PstI in the 2µ ori from pYES2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR
Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P35 template
0.5 µl	1:10 dilution of O42 (10 pmol/µL)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P35 template
0.5 µl	1:10 dilution of O43 (10 pmol/µL)
16.5 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	10	95°C	30 sec
		55°C	1 min
		67°C	6 min

• Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
• 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 on ice
• addition of 1 µl of the PCR product
• 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

Monday, 2th July

Repetition of PCR of PAL

Investigator: Mary

Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
4 µl	dNTP's (each 2.5 mM)
0.5 µl	Pfu Ultra II (2.5 U/µL)
5 µl	1:10 dilution of used forward primers (10µM)
5 µl	1:10 dilution of used reversed primers (10µM)
1 µl	DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL)
29.5 µL	bidest. sterile Water

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	2 min (and adding Pfu Ultra after 2 min)
2	30	95°C	30 sec
		55°C	1 min
		72°C	2.5 min
3		72°C	5 min

PCR of LexA with primers including the RFC25 pre- and suffix

Investigator: Jeffery Truong, Georg Schützinger

Aim of the experiment: The Biobrick BBa_K105005 (LexA) has a RFC10 pre- and suffix, but we need RFC25 pre- and suffix for protein fusion, so we have to do a PCR with primer containing the RFC25 pre- and suffix.

• The received forward and reverse primer TUM12-LexA-fw and TUM12-LexA-rv are resuspended in 204 µL and 221 µL ELGA water as described in the data sheet to get a final primer concentration of 100 pmol/µL=100 µM. For the PCR reaction mixture we took 0.5 µL of these resuspended primer and add 4.5 µL of ELGA water to get a final primer concentration of 10 µM.

• Clone 3 of BBa_K105005 (LexA) has beed choosen for the PCR (ERG No. P23).

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer
1 µl	10 µM Reverse Primer
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (BBa_K105005) from P23 (Clone 3)
35.75 µL	ELGA Water
=50 µL	TOTAL

• The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	55 °C	60 s
	68 °C	60 s
Final extension	68 °C	5 min
Hold	4 °C	overnight

Tuesday, 3th July

Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix

Investigator: Jeffery Truong

Aim of the experiment: Analytical gelelectrophoresis of cleaned up PCR product from LexA (BBa_K105005) with primer containing the RFC25 pre- and suffix (TUM12-LexA-fw and TUM12-LexA-rv).

• The clean-up of the PCR product LexA (BBa_K105005) with primer containing the RFC25 pre- and suffix (TUM12-LexA-fw and TUM12-LexA-rv) was performed with the QIAquick PCR Purification Kit from Qiagen after manufacturer's protocoll.

• 5 µL of the purificated PCR product was taken to perform a analytical gelelectrophoresis to verify the success of the PCR.

• 1% agarose gel containing ethidium bromide was used for the analytical gelelectrophoresis.

• The analytical gelelectrophoresis was performed for 60 min at 90 V.

• Scheme of the gel:

100 bp ruler

PCR product of BBa_105005

1000 bp ruler

• 

LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase

Investigator: Lara Kuntz

Aim of the experiment: To get colonies of BL21 strains containing lavendula LS for amplification and subsequent plasmid extraction.

• Schwab strain #106 was plated on a chloramphenicol containing LB plate. #108 was plated on a amp+chlp containing LB plate. The plates were incubated at 37°C over night.