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Team:Mexico-UNAM-CINVESTAV/Notebook/Week Four
From 2010.igem.org
Contents |
Week #4
27th September - 01 October 2010
Monday
Well we are stuck with the vector this week we have to get vector is our main objetive.
- We have achived a nanodrop readings as below
| ng/μl | 260/280 | 260/230 | |
| PsB1C3 | 0.60 | -0.73 | -0.0 |
| PCR 1 red | 427.70 | 1.71 | 1.71 |
| PCR 2 red | 483.20 | 1.74 | 1.71 |
| PCR 3 red | 410.70 | 1.76 | 2.02 |
| PCR 4 red | 431.05 | 1.61 | 1.88 |
| PCR 1 blue | 533.35 | 1.71 | 1.75 |
| PCR 2 blue | 536.00 | 1.72 | 1.82 |
| PCR 3 blue | 577.50 | 1.69 | 1.59 |
| PCR 4 blue | 627.55 | 1.70 | 1.56 |
| PsB1C3 | 4.10 | 2.62 | 1.01 |
- Yep our trouble is the lisis alcaline method to do the mini prep
we have a low concentration of vector’s plasmid DNA.
We have to work an try to get more plasmid and make dilutions of PCR’s
is not posible to do ligations with this ratio betwen vector an insert.
On this step we advisor has proposed a method via Low Meelting agarose
to geting out plasmid from the band.
Tuesday
We run with a low meelting's agarose prove to extract band tomorrow
we do a protocol to precipit with alchol an salts our objetive is do it all
to get vector and do the ligations.
Wensday
Plan
- We prepared all to do ligations PsB1C3 with our Pcr's.
- For this we going to cut with EcoRI and PstI both vector and insert.
- We'll run a low melting gel slow 2 hours then purified from the band.
At Claudia's lab we cut tree bands from 2% agarose gelof which two of
them where purified by Axigen kit the third one by a diferent kit.
The digestion was done with following amounts.
| DNA | 20μl |
| Buffer NB2 | 3μl |
| EcoRI | 2μl |
| PstI | 2μl |
| H2O | 2.4μl |
| BSA | 0.6μl |
| Total | 30μl |
