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• Use PCR Clean-up kit to clear the PCR product

This should result in 50ul of final product

• Measure the concentration of the product

This step will will be useful to determine how much of the product to use for the ligation

• Digest the PCR product (Check the restriction sites and replace EcoRI and SpeI as you need)
  • Prepare a final volume of 70ul using

- 50ul of the PCR product
- 3ul of EcoRI
- 3ul of SpeI
- 7ul of 10X buffer
- Use water to make the final volume 70ul

• • Mix everything well and incubate at 37C for an hour
  • Run the total 70ul on the gel using large wells
• Cut the gel fragment containing the DNA and extract it. The final volume will be 50ul.
• Run 5ul of extracted DNA on the gel to make sure it is right
• Ligate the extracted DNA with the plasmid backbone(e.g pSC1AT3) cut with the appropriate sites(E.g EcoRI and SpeI)
  • Prepare a final volume of 30ul using

- 3ul of ligation buffer(it is in the freezer)
- 1ul of ligase
- x (To be decided) ul of the backbone
- y (To be decided) ul of the insert
- Water totalling to 30ul

• • Leave the mix on the bench for three hours. To leave overnight suspend the eppendorf tube in a water box and place it in the fridge overnight
• Transform E.coli with the plasmid containing the insert
  • Use the total 30ul for the transformation
  • For + control use the plasmid without any insert
  • For - control use water to transform