Team:Heidelberg/notebook/rfc

iGEM Team Heidelberg 2015

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week number 37

▼2015-09-13 CPEC cloning of ATP-dependent Spinach12/13 and HHR-Malachite green into the RFC

Linearized RFC (1st September) and ATP-dependent Spinach (11th September) and HHR-Malachite green were assembled using Phusion Flash MM.

	cStock	cFinal	V[µL]
Phusion Flash MM	2x	1x	12.5
Linearized RFC	54 ng/µL	2.7 ng/µL	1.25
ATP-dep. Spinach12/13/ Malachite green-Insert	19.4/24.3/39.7 ng/µL		3.2/2,5/1
H2O		Ad 10	8.05/8.75/10.25
Final			25

 

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	5
Annealing/ Elongation	72	1 min
Final elongation	72	3 min	1
Hold	12	∞	1

 

• 2 µL of reaction were transformed into NEB 5-alpha Competent E.coli as described in the user’s manual and spread on a LB agar containing CM

week number 36

▼2015-09-01 Linearization of RFC 2015-09-01

RFC was linearized using xxfs044xx and xxfs045xx in a two step PCR using Phusion Flash MM.

	cStock	cFinal	V[µL]
Primer 1	100 mM	2 µM	2
Primer 2	100 mM	2 µM	2
RFC plasmid 1	150 ng/µL		1
DMSO		5 %	5
Phusion Flash Master mix	2 x	1 x	50
H2O		ad 100 µL	40
Final			100

PCR settings:

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	39
Annealing/ Elongation	72	30 s
Final elongation	72	3 min	1
Hold	12	∞	1

▼2015-09-01 Gibson assembly of Spinach and ATP-dependent Spinach into the RFC

Linearized RFC (1st September) and Spinach (25th August) and ATP-dependent Spinach (1st September) were assembled.

	cStock	cFinal	V[µL]
Gibson assembly MM	2x	1x	5
Linearized RFC	54 ng/µL	2.7 ng/µL	0,5
Spinach- | ATP-dep. Spinach-Insert	9.7/ 24 ng/µL	0.5/ 1.2 ng/µL | 1/ 2.4 ng/µL	0.5 | 1
H2O		Ad 10	4 | 3.5
Final			10

 

• Reaction was incubated for 30 min at 50 °C
• 2 µL of reaction were transformed into NEB 5-alpha Competent E.coli as described in the user’s manual and spread on a LB agar containing CM

▼2015-09-01 CPEC cloning of Spinach and ATP-dependent Spinach into the RFC

Linearized RFC (1st September) and Spinach (25th August) and ATP-dependent Spinach (1st September) were assembled using Phusion Flash MM.

	cStock	cFinal	V[µL]
Phusion Flash MM	2x	1x	12.5
Linearized RFC	54 ng/µL	2.7 ng/µL	1.25
Spinach- | ATP-dep. Spinach-Insert	9.7/ 24 ng/µL	0.5/ 1.2 ng/µL | 1/ 2.4 ng/µL	1.25 | 2.5
H2O		Ad 10	10 | 8.75
Final			25

 

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	5
Annealing/ Elongation	72	1 min
Final elongation	72	3 min	1
Hold	12	∞	1

 

• 2 µL of reaction were transformed into NEB 5-alpha Competent E.coli as described in the user’s manual and spread on a LB agar containing CM

▼2015-09-02 Primer extension for ATP-dependent Spinach

Primers xxms016xx and xxms017xx were extended using Phusion Flash MM.

	cStock	cFinal	V[µL]
Primer 1	100 mM	2 µM	2
Primer 2	100 mM	2 µM	2
DMSO		5 %	5
Phusion Flash Master mix	2 x	1 x	50
H2O		ad 100 µL	41
Final			100

PCR settings:

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	5
Annealing	62	30 s
Elongation	72	30 s
Final elongation	72	2 min	1
Hold	12	∞	1

▼2015-09-02 Picking colonies from ATP-dependent Spinach and Spinach II in pSB1C3 RFC for colony PCR and overnight culture

3 colonies were picked from each condition and check for insert in PCR. Same colonies were used to inoculate overnight cultures for Mini Prep

	cStock	cFinal	V[µL]
One Taq MM	2x	1x	5
Primer fwd			0.5
Primer rev			0.5
H2O		Ad 10	4
Final			10

 

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	32
Annealing	51	30 s
Elongation	68	1 min
Final elongation	68	3 min	1
Hold	12	∞	1

 

• 5 mL LB plus CM were inoculated with colony 1 to 6 an grown at 37 °C
• PCR products of colonies were checked on 1 % agarose gel and visualized with EtBr under UV light

Results & Outlook:

Cryo stocks will be prepared and Plasmid will be prepped.

▼2015-09-02 Cryostock and Miniprep of RFC

Cryostock:

500 µL of oN culture and 500 µL of autoclaved 40 % glycerol solution were mixed. Stock was stored at -80 °C.

Miniprep:

• Plasmid was purified using QIAprep Miniprep kit according to manufacturer’s protocol.
• An aliquot was sent to GATC for sequencing using xxmj006xx and xxmj07xx.
• Sequence was confirmed.

Outlook:

ivT will be performed: testing the three option that will be described in our RFC

week number 35

▼2015-08-25 RE-cloning: Digest of pSB1C3 containing RFP and Insert for RFC preparation

2 µg ofpSB1C3 and RFC preparation insert were incubated with EcoRI and SpeI 1 h at 37 °C with subsequent heat inactivation at 80 °C for 10 min.

	V[µL]
EcoRI	1
SpeI	1
Insert/pSB1C3	3/8
Cut Smart	5
H2O	40/35
Final	50

Digest was checked on 1% agarose in TAE gel and visualized with EtBr under UV light.

Insert was purified using Qiagen PCR purification Kit; pSB1C3 backbone was excised from 0,8 % agarose gel and purified using gele extraction kit from Qiagen.

▼2015-08-25 Primer extension for RFC insert plus RFC-Spinach

Oligos xxfs022xx and xxfs038xx (Spinach-Insert for RFC preparation) and xxfs046xx and xxfs047xx (Spinach for RFC) were annealed and extended using Phusion Flash:

	cStock	cFinal	V[µL]
Primer 1	100 mM	2 µM	2
Primer 2	100 mM	2 µM	2
DMSO		5 %	5
Phusion Flash Master mix	2 x	1 x	50
H2O		ad 100 µL	41
Final			100

PCR settings:

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	5
Annealing	62	30 s
Elongation	72	30 s
Final elongation	72	2 min	1
Hold	12	∞	1

Size of the samples were checked on a 1 % agarose in TAE gel and visualized with Ethidiumbromide (EtBr) under UV light (see gel picture).

▼2015-08-29 Ligation and trafo of pSB1C3 and RFC preparation insert

50 ng of pSB1C3 backbone was incubated with 5-fold excess of RFC-preparation insert with T4 DNA ligase in appropriate buffer at 37 °C for 1 h.

	V[µL]
T4 DNA Ligase	1
T4 DNA Ligase buffer	1
Insert	0.5
pSB1C3 backbone	2
H2O	5.5
Final	10

 

Trafo:

2 µL of Ligation were added to 40 µL of DH5alpha high efficiency from NEB. Cells were transformed as described in the manual.

▼2015-08-30 Picking colonies from RFC trafo for colony PCR and overnight culture

6 colonies were picked and check for insert in PCR. Same colonies were used to inoculate overnight cultures for Mini Prep

	cStock	cFinal	V[µL]
One Taq MM	2x	1x	5
Primer fwd			0.5
Primer rev			0.5
H2O		Ad 10	4
Final			10

 

	Temperature [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	32
Annealing	51	30 s
Elongation	68	1 min
Final elongation	68	3 min	1
Hold	12	∞	1

 

• 5 mL LB plus CM were inoculated with colony 1 to 6 an grown at 37 °C
• PCR products of colonies 1 to 6 are checked on 1 % agarose gel and visualized with EtBr under UV light

Results & Outlook:

Colonies 1 and 2 are positive according to fragment of colony PCR on agarose gel.

Cryo stocks will be prepared and Plamid will be prepped and test digested.

▼2015-08-30 Cryostock and Miniprep of RFC

Cryostock:

500 µL of oN culture and 500 µL of autoclaved 40 % glycerol solution were mixed. Stock was stored at -80 °C.

Miniprep:

• Plasmid was purified using QIAprep Miniprep kit according to manufacturer’s protocol.
• An aliquot was sent to GATC for sequencing using xxmj006xx and xxmj07xx.
• Sequence was confirmed.