Restriction enzyme : EcoRI , XbaI , SpeI , PStI

1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade
2. Put into Eppendorf (remember to keep the gel weight in 100-200mg)
3. Add equal volume of binding buffer , vortex
4. Incubate it until all the gel is dissolved in the buffer
5. Transfer all the solution to the purification column then centrifuge for 15,000rpm, 1min
6. Discard the flow-through and add 100μl binding buffer then centrifuge for 15,000rpm, 1min
7. Discard the flow through and add 700μl wash buffer then centrifuge for 15,000rpm, 1min
8. Discard the flow through and repeat (7) process
9. Discard the flow through and centrifuge for 15,000rpm, 7 min
10. Transfer the purification column to the 1.5ml eppendorf
11. Incubate it in 50-55℃ for 10 min
12. Add 20μl $H_2O$ (prewarm) or Elution buffer into the center of the column and wait for 2-3 min
13. Centrifuge for 15,000rpm, 7 min
14. Use nanodrop to quantify the nucleic acids’ concentration

1. Add 3 volumes of binding buffer, mix completely
2. Transfer the solution into purification column, centrifuge for 1 min in top speed
3. Discard the flow-through and add 700μl of wash solution, centrifuge for 1 min in top speed
4. Discard the flow-through and repeat step (3)
5. Centrifuge for 7 min in top speed
6. Incubate in 50-55℃
7. Add rewarmed $H_2O$ or elution buffer into the center of the column, wait for 2-3 min
8. Centrifuge for 7 min in top speed
9. Use nanodrop to quantify the nucleic acids’ concentration

1. Calcμlate the vector and insert ‘s ratio $${ng\ of\ vector\times\ kb\ size\ of\ insert\times molar\ ratio\ of\ insert\over kb\ size\ of\ vector\ vector} = ng\ of\ insert$$ vector : insert = 1 : 3 is quite optimal
2. Add 10X buffer
3. Add T4 ligase
4. Add $H_2O$ till 20μl in total volume
5. Put it in 4℃ overnight or 22℃ for 3 hr

1. Add 10μl DNA into 40 μl competent cell
2. Put it on the ice for 20-30min
3. 42℃ heat shock
4. Put it back onto the ice for 3 min
5. Add 250 SOC medium and 37℃ incubate for 1 hr
6. Plate all of the transformation and equally distribute it onto a 10 cm LB agar plate containing the appropriate antibiotic
7. 37℃ incubate overnight

1. 2–3 Fragment Assembly DNA 0.02-0.5 pmols, Gibson Assembly Master Mix 10μl, Add $H_2O$ to 20μl in total volume
2. Incubate in 50℃ for 15 min when 2-3 fragment assembly, store on ice or in -20℃ for transformation