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Revision as of 12:38, 4 October 2010 (view source) Mimmi (Talk | contribs) ← Older edit		Revision as of 10:25, 17 October 2010 (view source) Mimmi (Talk | contribs) (→Gel) Newer edit →
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	=== SOD.his / his.SOD / yCCS ===		=== SOD.his / his.SOD / yCCS ===

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Revision as of 10:25, 17 October 2010

Contents 1 Andreas 1.1 Preparation of Top10 chemically competent cells 1.2 pSB1C3.N-TAT & .N-Tra10 1.3 Assembly of new parts 1.3.1 Digestions 1.3.2 Ligations 1.3.3 Transformation 2 Mimmi 2.1 MITF-M 2.1.1 FB PCR 2.1.2 Digestion 2.1.3 Gel 2.2 SOD.his / his.SOD / yCCS

Andreas

Preparation of Top10 chemically competent cells

No growth on neither Amp 100, Cm 25 nor Km 50 plates - no contamination of competent cells.

pSB1C3.N-TAT & .N-Tra10

Successful transformation. One colony of each plate picked and dissolved in 10 μl and saved in fridge for later glycerol stock preparation.

Assembly of new parts

Planned and designed cloning strategies for some of the assemblies I will be constructing:

• pEX.SOD
• N-TAT⋅SOD⋅His
• N-Tra10⋅SOD⋅His
• N-LMWP⋅SOD⋅His (N-LMWP n/a)
• His⋅SOD⋅C-Tra10 (C-Tra10 n/a)
• His⋅SOD⋅C-TAT (C-TAT n/a)
• His⋅SOD⋅C-LMWP (C-LMWP n/a)
• RBS.yCCS

Digestions

• [pSB1C3.SOD] = 105.5 ng/μl
• [pSB1A2.RBS 34] = 60 ng/μl
• [pMA.His⋅SOD] = 266 ng/μl
• [pMA.SOD⋅His] = 246 ng/μl
• [pSB1C3.N-TAT] = 200 ng/μl
• [pSB1C3.N-Tra10] = 151 ng/μl
• [pSB1K3.RFP] = 104 ng/μl

	His⋅SOD	SOD⋅His	RBS 34	pSB1K3	N-TAT	N-Tra10	SOD
10X FD buffer	3	3	3	3	3	3	1
dH2O	16.9	17.5	0	5.8	15	11.8	7
DNA	8.1	7.5	25	19.2	10	13.2	1
NgoMIV (NEB)	1	0	0	0	0	0	0
PstI	1	0	1	1	0	0	1
FD SpeI	0	0	1	0	0	0	0
FD EcoRI	0	1	0	1	1	1	0
AgeI	0	1	0	0	1	1	0
FD XbaI	0	0	0	0	0	0	1
	30 μl	30 μl	30 μl	30 μl	30 μl	30 μl	10 μl

• Incubation: 37 °C
  • 2:00 (conventional enzymes)
  • 0:30 (FastDigest enzymes)
• Inactivation: 80 °C, 20 min

Ligations

• [Dig RBS 34 14/9] = 50 ng/μl
• [Dig pSB1K3 14/9] = 66.6 ng/μl
• [Dig SOD⋅His 14/9] = 66.6 ng/μl
• [Dig His⋅SOD 14/9] = 66.6 ng/μl
• [Dig N-TAT 14/9] = 66.6 ng/μl
• [Dig N-Tra10 14/9] = 66.6 ng/μl
• [Dig SOD 14/9] = 15 ng/μl

	Lig pEX.SOD 14/9	Lig pK.N-TAT⋅ SOD⋅His 14/9	Lig pK.N-Tra10⋅ SOD⋅His 14/9	Lig pA.RBS. yCCS 14/9
10X T4 ligase buffer	2	2	2	2
Vector DNA	3	1.5	1.5	2
Insert DNA (1)	10	11	11	6
Insert DNA (2)	–	4.5	4.5	–
dH2O	4	0	0	9
T4 DNA ligase	1	1	1	1
	20 μl	20 μl	20 μl	20 μl

• Incubation: 22 °C, 15 min

Transformation

Standard transformation

• 1 μl ligation mix
• 15 min on ice
• 50 μl IPTG (on pEX.SOD Amp 100 plate only)

Mimmi

MITF-M

FB PCR

mix	(µl)x2
sH2O	22.5		primers		Conditions
F primer	1		MITF_FB_F_18aug		time	°C
R primer	1		MITF_FB_R_18aug		10m	95
DNA	0.5				30s	95	)
tot	25µl				30s	55	> 5 cycles
					1m40s	72	)
					30s	95	\
					2m10s	72	/ 25 cycles
					10m	72

Digestion

- MITF-M and pSB1C3

Mix	MITF	pSB1C3 cut S	pSB1C3 cut E		conditions
sH2O	5	)	)		time	°C
DNA	20	>24	>24		30m	37
10xbuffer	3	)	)		20m	65
EcoRI	1	1
SpeI	1		1
tot	30µl	25µl	25µl

Gel

well	sample
1	ladder
2	MITF-M
3	MITF-M
4	MITF-M cut E+S
5	pSB1C3 cut E+S
6	pSB1C3 cut E+S

SOD.his / his.SOD / yCCS

• preparing for glycerol stock and over-expression...

SOD.his	x2	3ml
his.SOD	x2	3ml
yCCS	x2	3ml

• Grow at 37°C ON (~175rpm)