Will try regular pSB1A3 cut with XbaI and SpeI instead of dephosphorylated
Digestion
In a 30 μL reaction, cut a pSB1A2 plasmid (from Rene) XbaI and SpeI, then ran on 1% gel and used Zymoclean Gel DNA Recovery Kit
Reagent
pSB1A2
DNA
15.0
XbaI
1.0
SpeI
1.0
10x buffer
3.0
dH2O
10.0
Total vol.
30.0
Results of the enzyme digest is shown, with both pSB1A3 bands on the right. The gel was loaded with 30 μL samples. The bands that were cut are outlined in green.
Results from gel recovery
Sample
260
280
260/280
ng/μL
pSB1A3
0.007
0.004
1.711
6.916
Ligation
Use 20 ng of vector for each ligation = 3 μL pSB1A3
Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least 0.07 μL H2B
Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least 0.07 μL LOV
Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, the inserts were rounded to 1 μL for simplicity
H2B-LOV
Negative Control
H2B-LOV H2B insert DNA (1x mol vector)
1.0 μL
0
H2B-LOV LOV insert DNA (1x mol vector)
1.0 μL
0
Vector DNA (39 ng)
3.0
3.0
Gibson master mix
15.0
15.0
Total volume
20.0
18.0
Mix the reaction(s) thoroughly by flicking the tube. Incubate at 50°C for 1 hour.