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Revision as of 15:26, 26 October 2010 (view source) NinaSchiller (Talk | contribs) (→Ligation) ← Older edit		Latest revision as of 21:53, 27 October 2010 (view source) JohanNordholm (Talk | contribs) m
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	I incubated the ligations in a water bath, 22 °C in 15 min.		I incubated the ligations in a water bath, 22 °C in 15 min.
		+	==Johan==
		+
		+	===MIniprep===
		+	Two his-bFGF and two bFGF-his samples.
		+
		+	All ~400 ng/µl
		+
		+	===Digestion===
		+
		+	2 µl DNA
		+	2 µl 10x fastbuffer
		+	(1 µl BamHI)
		+	15 µl H2O
		+
		+	===Gel===
		+
		+	Cut & uncut
		+
		+	[[Image:SU 27sepgel.png]]
		+
		+	Results: All showed good results
		+
		+	===Cut bFGF-his===
		+
		+	3 µl DNA
		+
		+	1 µl NgoMIV
		+
		+	1 µl PstI
		+
		+	2 µl 10x fastbuffer
		+
		+	13 µl H2O
		+
		+	Then heat-inactivation
		+
		+	===Ligation===
		+
		+	Of cut bFGF-his into already cut TAT_N vector
		+
		+	5 µl bFGF
		+
		+	1 µl vector
		+
		+	2 µl 10x fastbuffer
		+
		+	1 µl T4 ligase
		+
		+	11 µl H2O
		+
		+	1h 37 °C
		+
		+	===Transformation
		+
		+	3 µl of all constructs was transformed into top10 cells
	{{Stockholm/Footer}}		{{Stockholm/Footer}}

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Latest revision as of 21:53, 27 October 2010

Contents 1 Andreas 1.1 Cloning and assembly 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.2 Sequencing 2 Mimmi 2.1 Over expression 3 Nina 3.1 Sequencing 3.2 Digestion 3.3 Concentration measurement 3.4 Ligation 4 Johan 4.1 MIniprep 4.2 Digestion 4.3 Gel 4.4 Cut bFGF-his 4.5 Ligation

Andreas

Cloning and assembly

Transformation results

From 25/9 transformations

Good colony yield on all plates. Good white:red colony ratio on pEX plates (Constructs 1, 2, 3 and 4).

Colony PCR

Picked colonies for colony PCR.

1. pEX.N-LMWP⋅SOD⋅His (K): 1-2
2. pEX.N-TAT⋅SOD⋅His: 1-2
3. pEX.N-Tra10⋅SOD⋅His: 1-2
4. pEX.N-LMWP⋅SOD⋅His (C): 1-2
5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1-4
6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS: 1-4
7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS: 1-4
8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1-4
9. BL21 pEX.N-TAT⋅SOD⋅His 3: 1-2
10. BL21 pEX.N-TAT⋅SOD⋅His 4: 1-2

Standard colony PCR protocol.

• Elongation time: 2:00

Gel verification

Gel 1
1 % agarose, 110 V

Gel 2
0.8 % agarose, 90 V

Expected bands

1. 744 bp
2. 735 bp
3. 765 bp
4. 744 bp
5. 1645 bp
6. 1636 bp
7. 1666 bp
8. 1645 bp
9. 735 bp
10. 735 bp

Results
Gels run too far (no data). New gels will be run tomorrow.

Sequencing

Samples prepared 25/9 were sent for sequencing. Sequencing information added to the 25/9 notebook page.

Mimmi

Over expression

Start cultures

*3ml LBAMP + tip from glycerol stock

*Grow ON in 37°C, 225rpm

DNA

pEX.SOD

pEX.yCCS

pEX.SOD.his

pEX.his.SOD

Nina

Sequencing

I send two samples for sequencing. 15 ul sample and 1.5 ul Forward primer.

• pMa #3 Tyrosinase_N ASB0045 694
• Tyrosinase in bank vector K ASB0045 695

Digestion

Digestion of Fusion (1/9):

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme NgoMIV 1 ul
• Restriction enzyme SpeI 1 ul (Add after 1.5 h in 37 °C & incubate in additional 30 min)

Digestion of Fusion (1/9):

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme AgeI 1 ul
• Restriction enzyme EcoRI 1 ul (Add after 1.5 h in 37 °C & incubate in additional 30 min)

Inactivate 20 min 80 °C all samples

Digestion of IgG #5_E_pMa:

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme NgoMIV 1 ul
• Restriction enzyme PstI 1 ul (Add after 1.5 h in 37 °C & incubate in additional 30 min)

Digestion of IgG #5_E_pMa:

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme AgeI 1 ul
• Restriction enzyme EcoRI 1 ul (Add after 1.5 h in 37 °C & incubate in additional 30 min)

Digestion of Protein A#5_E_pMa:

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme AgeI 1 ul
• Restriction enzyme EcoRI 1 ul (Add after 1.5 h in 37 °C & incubate in additional 30 min)

Digestion of Protein A#5_CPPs_N:

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme XbaI 1 ul
• Restriction enzyme PstI 1 ul

Incubate in 37 °C for 30 min.

Concentration measurement

I measured conc of the samples for preparation of the following ligation.

Ligation

The ligation was according to:

I incubated the ligations in a water bath, 22 °C in 15 min.

Johan

MIniprep

Two his-bFGF and two bFGF-his samples.

All ~400 ng/µl

Digestion

2 µl DNA 2 µl 10x fastbuffer (1 µl BamHI) 15 µl H2O

Gel

Cut & uncut

Results: All showed good results

Cut bFGF-his

3 µl DNA

1 µl NgoMIV

1 µl PstI

2 µl 10x fastbuffer

13 µl H2O

Then heat-inactivation

Ligation

Of cut bFGF-his into already cut TAT_N vector

5 µl bFGF

1 µl vector

2 µl 10x fastbuffer

1 µl T4 ligase

11 µl H2O

1h 37 °C

===Transformation

3 µl of all constructs was transformed into top10 cells