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

From OpenWetWare

Jump to: navigation, search
Hydrobium etli Main project page
Previous entry      Next entry

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.

Image:HydG_DD_aislado_concentracion.JPG

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

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.


Personal tools