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Revision as of 23:07, 25 October 2010 (view source) Mimmi (Talk | contribs) ← Older edit		Latest revision as of 11:04, 26 October 2010 (view source) Hassanforoughiasl (Talk | contribs)
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	==Andreas==		==Andreas==
	===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX===		===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX===
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Latest revision as of 11:04, 26 October 2010

Contents 1 Andreas 1.1 Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX 1.1.1 Colony PCR 1.1.2 Gel verification 2 Mimmi 2.1 Over expression 2.1.1 Gel

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Continued from 30/8 transformations

Colony PCR

1. BL21 pEX.nLMWP⋅SOD⋅His: A & B †
2. BL21 pEX.nTra10⋅SOD⋅His: A & B †
3. pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
4. pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
7. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
8. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

† From "Transformation of BL21, 30/8"

Standard colony PCR settings.

• Elongation time: 2:00

Gel verification

0.8 % agarose, 100 V

Expected bands:

1. 744 bp
2. 765 bp
3. 1523 bp
4. 1523 bp
5. 1553 bp
6. 1553 bp
7. 1532 bp
8. 1532 bp

Results
Ran gel a little too long too see correct clones of constructs 1 and 2. For the other it looks like many clones have double inserts, probably a result of too high insert:vector ratio during ligation.

Confirmed clones:

1. -
2. -
3. A
4. B
5. -
6. -
7. B
8. B

These will be saved for later.

Mimmi

Over expression

• Load PhastGel

Gel

well	sample		well	sample
1	ladder		1	ladder
2	SOD 0h		2	SOD.his 3h 1:4
3	SOD 50min		3	SOD.his sup
4	SOD 3h 1:4		4	his.SOD 0h
5	SOD sup		5	his.SOD 3h 1:4
6	SOD.his 0h		6	his.SOD sup