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	==15-08-2013==		==15-08-2013==
	===Gibson Assembly and electroporation===		===Gibson Assembly and electroporation===

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Latest revision as of 17:16, 2 October 2013

Contents 1 15-08-2013 1.1 Gibson Assembly and electroporation 1.1.1 Assembly of our fragments 1.1.2 Gibson backbone (pSB4K5) 1.1.3 Electroporation 2 16-08-2013 2.1 Colony PCRs 3 17-08-2013 3.1 Colony PCRs 3.1.1 Electroporation 4 18-08-2013 4.1 Colony PCRs

15-08-2013

Gibson Assembly and electroporation

Assembly of our fragments

Those gel-purified fragments were added for the Gibson assembly of our construct pFSN.

Fragment	Primer	Date	Concentration (ng/µl)	Volume (µl)
pSB4K5	FS_01 - FS_16	14-08-13	13	1.35
AF	FS_02 - FS_05	13-08-13	49.75	2.65
FG	FS_21 - FS_26	07-08-13	24.5	2.69
G	FS_35_SR_01 - FS_23	08-08-13	34.9	2.1
OP	FS_22 - FS_13	13-08-13	36.3	0.89
L	FS_14 - FS_15	07-08-13	51	0.31
Gibson MasterMix NEB			2x	10

• Gibson Assembly according to protocols of NEB, 1h at 50°C

Gibson backbone (pSB4K5)

The backbone pSB4K5 was also incubated with the Gibson mastermix. We will use this as one negative control in the electroporation to test the efficiency.

Fragment	Date	Concentration (ng/µl)	Volume (µl)
pSB4K5	14-08-13	13	1.35
H2O			8.65
Gibson MasterMix NEB		2x	10

• Gibson Assembly according to protocols of NEB, 1h at 50°C

Electroporation

The following samples were electroporated

mixture		volume used	cells
5 µl Gibson Assembly of pFSN construct	10 µl H2O	1 µl	50 µl DH10β old
5 µl Gibson Assembly of pFSN construct	10 µl H2O	14 µl	50 µl DH10β old
5 µl Gibson Assembly of Backbone (control)	10 µl H2O	1 µl	50 µl DH10β old
pSB4K5 14-08-13		1.35 µl	50 µl DH10β old
pSB6A1 Hanna [69.57 ng/µl] 1:10		1 µl	50 µl DH10β Fanny new

• Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  
• incubation and growth of cells for 1 hour at 37°C
  
• cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
• cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN and on agar plates containing ampicillin for electroporation of the control backbone pSB6A1

20130816ElectroporationA.JPG 1 µl of diluted Gibson electroporated; 10 µl of E.coli plated	20130816ElectroporationB.jpg 1 µl of diluted Gibson electroporated; Rest of E.coli plated	20130816ElectroporationC.jpg 14 µl of diluted Gibson electroporated; 10 µl of E.coli plated	20130816ElectroporationD.jpg 14 µl of diluted Gibson electroporated; Rest of E.coli plated
P1010689.JPG 1 µl of diluted Gibson (backbone control) electroporated; 10 µl of E.coli plated	P1010690.JPG 1 µl of diluted Gibson (backbone control) electroporated; Rest of E.coli plated	P1010692.JPG 1 µl of backbone control electroporated; 10 µl of E.coli plated	P1010693.JPG 1 µl of backbone control electroporated; Rest of E.coli plated
P1010704.JPG Test of electrocompetence DH10β with 1 µl pSB6A1; 10 µl of E.coli plated	P1010705.JPG Test of electrocompetence DH10β with 1 µl pSB6A1; Rest of E.coli plated

16-08-2013

Colony PCRs

As a lot of colonies grew we screened if DelL and pSB4K5 are present.

Different probes:

• A: 1 µl of diluted Gibson electroporated; 10 µl of E.coli plated
• B: 1 µl of diluted Gibson electroporated; Rest of E.coli plated
• C: 14 µl of diluted Gibson electroporated; 10 µl of E.coli plated
• D: 14 µl of diluted Gibson electroporated; Rest of E.coli plated

• Only red colonies were chosen for screening
• The expected amplicon size is 1.5 kb

Reaction mixture

Reagent	A1, A2	B1-B5	C1-C3	D1-D40
VR-Primer: (1/10)	2 µl	2 µl	2 µl	2 µl
FS_14: (1/10)	2 µl	2 µl	2 µl	2 µl
Colonies	2 red colonies A (A1, A2)	5 red colonies B (B1-B5)	3 red colonies C (C1-C3)	40 red colonies D (D1-D40)
DreamTaq	10 µl	10 µl	10 µl	10 µl
dd H2O	5 µl	5 µl	5 µl	5 µl

PCR Conditions

--> T100

Cycles-PCR	temperature [°C]	Time [s]
1	95	2:00
12	95	1:00
	66 ↓ 0.5	5
	72	1:30 min
18	95	1:00
	63	5
	72	1:30 min
1	12	inf

Result:

• None of the screened colonies led to the correct amplicon, therefore the correct assembly has not worked in any of the white colonies
• screening will be repeated, with white colonies as template, as bacteria including the correct 32 kbp backbone might not be capable of expressing mRFP anymore due to the large insert between lac promotor and mRFP

17-08-2013

Colony PCRs

Different probes:

• A 1 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
• B 1 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
• C 14 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
• D 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated

• Only white colonies where chosen for screening
• The expected amplicon size is 1.5 kb

Reaction mixture

Reagent	B1w	C1w	D1w-D10w
VR-Primer: (1/10)	2 µl	2 µl	2 µl
FS_14: (1/10)	2 µl	2 µl	2 µl
Colonies	1 white colony B (B1w)	1 white colony C (C1w)	10 white colonies D (D1w-D10w)
DreamTaq	10 µl	10 µl	10 µl
dd H2O	5 µl	5 µl	5 µl

PCR Conditions

--> T100

Cycles-PCR	temperature [°C]	Time [s]
1	95	2:00
12	95	1:00
	66 ↓ 0.5	5
	72	1:30 min
18	95	1:00
	63	5
	72	1:30 min
1	12	inf

Result:

• Colony D8w shows expected amplicon of 1.5kbp
• The other colonies were screened negative.
  
• D8w will be transfered to liquid culture to repeat screening PCR as well as for further validation beeing screening for the other insert fragments as well as test restriction digest
• Furthermore another electorporation will be carried out to yield more clones positive for screening with the Primers VR and FS_14, therefore the remaining 10µl of the Gibson assembly will be purified using isopropanol precipitation

Electroporation

We electroporated the Gibson assembly (15-08) again to increase the chance of a positive clone. We purified it by isopropanol purification.

mixture		volume used	Cells
Assembled fragments (15-08) isopropanol precipitated		15 µl	50 µl DH10β

• Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  
• incubation and growth of cells for 1 hour at 37°C
  
• cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
• cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN

P1010701.JPG 15 µl of Gibson 15-08 (isopropanol precipitated) electroporated; 10 µl of E.coli plated

P1010702.JPG 15 µl of Gibson 15-08 (isopropanol precipitated) electroporated; Rest of E.coli plated

18-08-2013

Colony PCRs

Different probes:

• D: 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
• E: 1 µl isopropanol precipitated Gibson Assembly (15-08) ; 10 µl of E.coli plated
• F: 1 µl isopropanol precipitated Gibson Assembly (15-08); remaining E.coli plated

• The expected amplicon size is 1.5 kb
• 3 colonies were sceened in one PCR
  

Reaction mixture

Reagent	D8w	D11w-D24w	E1w-E19w	F1w-F20w	E1r-E10	F1r-F20r
VR-Primer: (1/10)	2 µl	2 µl	2 µl	2 µl	2 µl	2 µl
FS_14: (1/10)	2 µl	2 µl	2 µl	2 µl	2 µl	2 µl
Colonies	Colony D8w liquid culture	24 white colonies D (D1w-D24w)	19 white colonies E (E1w-E19w)	30 white colonies F (F1w-F20w)	10 red colonies E (E1r-E10r)	20 red colonies F (F1r-F20r)
DreamTaq	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
dd H2O	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl

PCR Conditions

--> T100

Cycles-PCR	temperature [°C]	Time [s]
1	95	2:00
12	95	1:00
	66 ↓ 0.5	5
	72	1:30 min
18	95	1:00
	63	5
	72	1:30 min
1	12	inf

Result:

• colony D8w again showed the expected amplicon in the colony PCR and will therefore be further analyzed for the other fragments as well as restriction digested and partially sequenced as soon as screening primers arive
• Furthermore colonies E16w and F25w are screened positive for the fragment DelL