Summary
Digestion/ ligation reaction
-
Dilute the purified PCR product to 20 fmol/μL
- Measure ng/μL of the purified sample.
- The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
- Formula: x = length in bp ÷ measured ng/μL * 0.26
Purified PCR Product to 20fmol/μL
| Sample
| μL of Sample
| μL of dH2O
|
| 1. pSB1A3 | 7.8 | 12.2
|
| 2. hPCD | 2.8 | 17.2
|
| 3. BL02 | 6.4 | 13.6
|
| 4. BL03 | 5.5 | 14.5
|
| 5. BL04 | 8.8 | 11.2
|
| 6. fshPCD | 2.3 | 7.7
|
- Perform BsmBI/ T4 ligase mediated assembly
- BsmBI cuts the DNA fragments and creates complementary overhangs.
- Complementary sticky ends anneal via base pairing.
- T4 ligase seals gaps in the phosphodiester DNA backbone.
| Reagent
| Vol.
| Thermal cycling
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
|
| 20 fmol of each DNA part | up to 8.0
|
| 10x T4 ligase buffer (Promega) | 1.0
|
| T4 ligase (NEB) | 0.25
|
| BsmBI | 0.5
|
| dH2O | 0.25
|
| | 10.0 μL
|
|
Engineering PC-TFs
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