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

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====Concentration calculation====
====Concentration calculation====
*HydG = 0.0006 pM/μL (defined in the previous gel)
*HydG = 0.0006 pM/μL (defined in the previous gel)
*pSB1C3 = 0.012 pM/μL (defined by [[User:Gustavo_A._Ruiz_Buendia|Gustavo]] in [http://openwetware.org/wiki/IGEM:UNAM_LCG/2009/Notebook/Hydrobium_etli/2011/09/23 this gel]
*pSB1C3 = 0.012 pM/μL (defined by [[User:Gustavo_A._Ruiz_Buendia|Gustavo]] in [http://openwetware.org/wiki/IGEM:UNAM_LCG/2009/Notebook/Hydrobium_etli/2011/09/23 this gel])
*Relation: 0.6:12
*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
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]
*Final volume and final concentration: 18[0.2]
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*X[0.12]=18[0.2]
*X[0.12]=18[0.2]
*Final disolution: X = 0.3μL pSB1C3 + 17.7μL H<sub>2</sub>O
*Final disolution: X = 0.3μL pSB1C3 + 17.7μL H<sub>2</sub>O
====Ligation details====
I made the ligations with [http://www.neb.com/nebecomm/products/productm0202.asp 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====
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;" <!-- This line here formats your table for you.  Change the code to change the formatting of your table.-->
| align="center" style="background:#f0f0f0;"|'''Reactive'''
| align="center" style="background:#f0f0f0;"|'''Ammount (μL)'''
<!-- Copy and paste one of the lines above to create a new column in the schedule table.  Alternatively, you can also delete lines to reduce the number of columns.-->
|--
| HydG (insert)
| 8.5
|--
| pSB1C3 (vector, dilution)
| 8.5
|--
| Ligase buffer 10X
| 2
|--
| T4 ligase
| 1
|--
| '''Total'''
| '''20'''
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.-->
|}




Revision as of 22:51, 24 September 2011

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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)
  • 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



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