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Revision as of 15:34, 24 October 2010 (view source) NinaSchiller (Talk | contribs) (→Nina) ← Older edit		Revision as of 15:45, 24 October 2010 (view source) NinaSchiller (Talk | contribs) (→Andreas) Newer edit →
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		+	==Nina==
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		+	===Agarose gel on Colony PCR===
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		+	I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.
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		+	[[Image:Aq1.jpg|200px]]
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		+	[[Image:Aq2.jpg|200px]]
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		+	[[Image:Aq3.jpg|200px]]
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		+	[[Image:Aq4.jpg|200px]]
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		+	Unfortunately non of these gels show that there has been an proper insert of Fusion and Protein A all with the three CPPs in the N-terminal in the pEX vector.
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		+	I made a bad decision to conduct the ligation of these parts with the pEX vector and transform the ligations directly into overexpression cells such as BL21. I should have transformed the ligations into cloning cells such as Top 10 and from there as usual perform a mini prep and transform the vectors with insert into BL21 cells. However I hoped some of the ligations that would have ended up as vectors with insert would succesfully get transformed into BL21, but it seems from the gels that this has not happened. We are short of time in the competition and this was a test to see if I could skip the two days I had to spend with transformation into Top 10, but it unfortunatelly did not work out this time.
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	==Andreas==		==Andreas==

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Revision as of 15:45, 24 October 2010

Contents 1 Nina 1.1 Agarose gel on Colony PCR 2 Andreas 2.1 Colony PCRs 2.1.1 Gel verification 2.2 PCR for Uppsala team 2.2.1 Gel verification

Nina

Agarose gel on Colony PCR

I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.

Unfortunately non of these gels show that there has been an proper insert of Fusion and Protein A all with the three CPPs in the N-terminal in the pEX vector.

I made a bad decision to conduct the ligation of these parts with the pEX vector and transform the ligations directly into overexpression cells such as BL21. I should have transformed the ligations into cloning cells such as Top 10 and from there as usual perform a mini prep and transform the vectors with insert into BL21 cells. However I hoped some of the ligations that would have ended up as vectors with insert would succesfully get transformed into BL21, but it seems from the gels that this has not happened. We are short of time in the competition and this was a test to see if I could skip the two days I had to spend with transformation into Top 10, but it unfortunatelly did not work out this time.

Andreas

Colony PCRs

• pEX.ProtA⋅His: PA 1 & 2
• pEX.IgGp: Ig 1 & 2

Standard colony PCR settings

• Elongation time: 1:20

Gel verification

Expected bands

• PA: 417 bp
• Ig: 1149 bp

Results
All four constructs confirmed.

PCR for Uppsala team

Gel verification

Expected bands

• All constructs: ≈ 6570 bp

Results
Incorrect size. Send to the Uppsala team for analysis.