IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/24
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Now that we have HydG digested and purified we need to put it into the vector pSB1C3 to send it to the iGEM.
I first ran a gel to see HydG's concentration and then I calculate the ammounts of pSB1C3 to make the ligation. Unfortunatly it looks like I lost a lot of HydG during the band extraction so the ligation was done with very little ammounts of DNA.
I ran this gel to know HydG's concentration.
1.HydG 2.Ladder fermentas The rest of the lines are not of interest for this analysis.
The concentration is VERY low, the mark at 1700bp aprox is barely visible. Gustavo helped me to calculate the concentration, we decided that it is 0.003pM
We need to do this ligation to send HydG it to the registry
Since we need a 3:1 cassete:vector relation we want a 0.2 vector concentration to get 0.6:0.2 relation.
I made the ligations with T4 DNA ligase by NEB™.
I made one ligation reaction and one control reaction in which I sustitute HydG for water. The ligation should go well because the enzimes EcoRI and PstI (used to cut HydG and pSB1C3) left sticky ends.