EPF-Lausanne/11 August 2008

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=Molecular Biology= We try by different ways to assemble biobricks

We(Vincent) did a mistake with the kanamycin stock, we put 1000 time less the dose.

We start a cell culture of all interesting biobricks so we can do a new mini-prep tomorrow, because the mini-prep that we use are not good(kanamycin 1000 time less concentrated).

E1010 and F1610
We tried last week to assemble two big parts because it is easier to purify them. We did the ligation and transformation. The result was great, all of the ten plates worked. Today, we decide to do a mini-prep of these cells to purify the DNA. Then we want to check the plasmid so we digest a small quantity of them with EcoRI and SpeI to prepare it for a gel tomorrow.

by Gel
We try again purifying insert and vector usign a gel.

We try to assemble :

R0040/B0034

R0071/B0034

and I1466/I1433

We have successfully isolated from the gel : R0040, R0071 and B0034

We try changing strategy to isolate I1466 and I14033.

I1466/I14033
I14033 is small. We keep it in the vector. I1466 is big we use it as an insert. We cut with SpeI and PstI. We digest the plasmid and we are going to do the ligation tomorrow.

Without gel, use PCR with biobrick primer
Tomorrow, we plan on using the new mini-preps to amplify DNA by PCR, this way we don't need to use gel purification (which is hard to do with small fragments).

Addition : We still need gel purification after we digest the PCR product, so that enzymes and other fragments are removed. However this way we use less of our mini-prep material.

http://openwetware.org/wiki/Knight:Annealing_and_primer_extension_with_Klenow_polymerase