"
Rensselaer/15 September 2008
From 2008.igem.org
Ligation of Fe promoter and ZntA promoter to deP mRFP vector.
Ratio is 3 insert to 1 vector
approximate sizes
2kb vector
200 bp ZntA
500 bp Fe
need 200 ng total:
3x concentration of insert DNA.
for ZntA ligation:
200 ng * 0.6/2.6 = 46 nanograms Znta 46 ng * 1 uL/7.6ng ~ 6.00 uL ZntA insert 200 * 2/2.6 = 153 nanograms vector 153(1 uL/30.3 ng) = 5 uL vector
process:
add water
then buffer then DNA then enzyme
Total Volumes
4 uL H20
5 uL dP-mRFP vector
6 uL ZntA insert
1 uL T4 DNA ligase
4 uL 5x ligase buffer
_____________________
20 uL total
Fe
2+1.5 = 3.5
200*1.5/3.5 = 85 ng Fe (1 ng/ 30 uL) = 3 uL Fe 200*2/3.5 = 115 vector (1 ng/ 30.3 uL) = 4 uL vector dP-mRFP
6 uL H20
4 uL vector mRFP
3 uL insert Fe
1 uL T4 DNA ligase
4 uL 5x ligase buffer
______________________
20 uL total
using 115 mRFP as negative control
11 uL H20
4 uL vector
1 uL T4 DNA ligase
4 uL 5x ligase buffer
______________________
20 uL total
