Rensselaer/15 September 2008

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