IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/24

Hydrobium etli

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HydG ligation Abstract 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. Gel Details I ran this gel to know HydG's concentration. 120V, 40min. 5μL HydG + 2μL dye; 2μL ladder Gel image Lanes: 1.HydG 2.Ladder fermentas The rest of the lines are not of interest for this analysis. Gel 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 HydG-pSB1C3 ligation We need to do this ligation to send HydG it to the registry Concentration calculation HydG = 0.0006 pM/μL (defined in the previous gel and with this DNA molarity calculator) pSB1C3 = 0.012 pM/μL (defined by Gustavo in this gel) Relation: 0.6:12 Since we need a 3:1 cassete:vector relation we want a 0.2 vector concentration to get 0.6:0.2 relation. Final volume and final concentration: 18[0.2] Needed volume and actual concentration: X[0.12] X[0.12]=18[0.2] Final disolution: X = 0.3μL pSB1C3 + 17.7μL H2O Ligation details 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. 14°C, 12hrs. Ligation ammounts Reactive Ammount (μL) HydG (insert) 8.5 pSB1C3 (vector, dilution) 8.5 Ligase buffer 10X 2 T4 ligase 1 Total 20 Note: in the control reaction I substituted 8.5μL of HydG for 8.5μL of water.