EPF-Lausanne/13 August 2008

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=Molecular biology=
=Molecular biology=
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We do great mini-prep.
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Yesterday's cell cultures and mini-prep worked out fine.
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==F1610/E1010==
==F1610/E1010==
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We do the gel of F1610 and E1010 from old mini-preps that we let digest all the night by SpeI and EcoRI. E1010 is great but F1610 doesn't work.
 
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We don't know where come the mistake!!!
+
We do the gel of F1610 and E1010 from the old mini-preps that we digested with SpeI and EcoRI all night. E1010 is great but F1610 doesn't work.
-
So we want to be sure that the part are correct and enzymes too. So we digest F1610 and E1010 from new mini-prep of today and we split experiences of digest in two EcoRI/SpeI and PstI/XbaI.
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We don't know where the mistake comes from.
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And we do different PCR by this way we don't use restriction enzyme. We do PCR with biobrick primer for E1010, F1610, F1610/E1010(assembled) and we do a positive control with DNA and Primers form Bart.
+
We want to be sure that the part and the enzymes are fine, so we digest F1610 and E1010 from the new mini-preps we did today, doing two experiences using both combination of enzymes: EcoRI/SpeI and PstI/XbaI.
-
In finally we will run a gel with :
+
We also do a PCR, this way we don't use restriction enzymes. We do the PCR with Biobrick primers (Prefix and suffix) for E1010, F1610, F1610/E1010(presumably assembled) and we do a positive control using DNA and Primers Bart gave us.
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Finally we run a gel with :
F1610 digest by EcoRI and SpeI
F1610 digest by EcoRI and SpeI
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E1010 from PCR
E1010 from PCR
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E1010/F1610 from PCR
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===Results===
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Restriction enzymes seem to be working fine!
 +
 
 +
E1010/F1610 from PCR (2 times from different colonies)
Positiv control from PCR
Positiv control from PCR
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F1610 (nothing, straight from mini-prep)
F1610 (nothing, straight from mini-prep)
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==I1466/I14033==
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The gel purification doesn't work. We don't see the insert, I1433. We thus decide to focus on E1010 and F1610.
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==R../B0034==
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We focus on E1010 and F1610.
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mini-prep of R../B0034
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PCR of minprep
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I1466/I14033 gel purification
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Latest revision as of 09:32, 19 August 2008

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Contents

Molecular biology

Yesterday's cell cultures and mini-prep worked out fine.

F1610/E1010

We do the gel of F1610 and E1010 from the old mini-preps that we digested with SpeI and EcoRI all night. E1010 is great but F1610 doesn't work.

We don't know where the mistake comes from.

We want to be sure that the part and the enzymes are fine, so we digest F1610 and E1010 from the new mini-preps we did today, doing two experiences using both combination of enzymes: EcoRI/SpeI and PstI/XbaI.

We also do a PCR, this way we don't use restriction enzymes. We do the PCR with Biobrick primers (Prefix and suffix) for E1010, F1610, F1610/E1010(presumably assembled) and we do a positive control using DNA and Primers Bart gave us.

Finally we run a gel with :

F1610 digest by EcoRI and SpeI

E1010 digest by EcoRI and SpeI

F1610 digest by PstI and XbaI

E1010 digest by PstI and XbaI

F1610 from PCR

E1010 from PCR

Results

Restriction enzymes seem to be working fine!

E1010/F1610 from PCR (2 times from different colonies)

Positiv control from PCR

E1010 (nothing, straight from mini-prep)

F1610 (nothing, straight from mini-prep)

I1466/I14033

The gel purification doesn't work. We don't see the insert, I1433. We thus decide to focus on E1010 and F1610.

R../B0034

We focus on E1010 and F1610.

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