Team:UNICAMP-Brazil/Notebooks/October 2

Preparation of electrocompetent E. coli

 * The efficiency test of the electrocompetent E. coli didn´t work. Probably the antibiotic concentration in the plates is wrong.

Taís

New Strategy: pGEM

 * This evening we had a wonderful idea to try to solve our problem in screening the new biobricks in LB plates. While we wait the new primers to arrive, we decided to use pGEM T-easy vector as a way to add the EcoRI restriction site in the PCR products that already have XbaI and SpeI sites. This strategy will permit the part to be ligated to the biobrick vector avoiding unwanted ligations between the cohesive ends and consequently the hard screening we were performing. Click here to see the hole strategy.


 * For detailed information about pGEM T-easy vector system, see the Manual.


 * So today we started digesting both PCR products and pGEM vector using SpeI. We believe that this digestion will facilitate the correct insertion of XbaI end besides EcoRI site. After digesting, we did the ligation reactions, transformed and plated it in LB+AMP+X-gal medium. We hope that tomorrow we will find few blue colonies and a LOT of white ones! =]

Gleidson and Taís

Customizing the PCR

 * Jen1 (ORF) band purification using the gel made yesterday and, after, we performed a PCR of this product to amplify the Jen1 (ORF).
 * Then we did a digestion of the Jen1(ORF) with the enzymes XbaI e SpeI.


 * PCR of the END part using the primers VF and VR.

Wesley

Problems on assembling our devices to be inserted into genomic DNA

 * Without the availability of BBa_J61000 and BBa_E0840, assembling those Upstream and Downstream contructions to be inserted into genomic DNA became really difficult, making us thinking how necessary and urgent they really are...
 * Seeing that, we decided to leave them aside and, for what may be the thousandth try, we will again focus on finO and finP biobricks construction, now using that new strategy with pGEM described by Gleidson and Taís.

Marcelo

Cre-Recombinase + pSB1A3 ligation

 * After successfully digesting both insert and vector with XbaI and SpeI restricion enzymes, we performed today the ligation between them, according to Protocol 11.
 * As we purified both sequences, we really expect an correctly ligation this time, hence resulting in no religation between the vector and it's part (RFP device).

Víctor

Cre-Recombinase Parallel Strategy

 * As we decided to try making our biobricks constructions with pGEM strategy for almost every construction we will need, Cre-Recombinase will also follow this same strategy as a parallel one, along with the standard one.
 * Therefore, we will try to construct Cre's Biobricks using two different strategies simultaneasly.

Victor

Kamikaze’s Minipreps

 * We did the miniprep with the innoculum made yesterday using the Protocol 2.

Marcos