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Contents 1 Steps to use the PCR products 1.1 PCR Clean-up 1.2 Digest the PCR product 1.3 Extract the DNA from the gel 1.4 Ligate the PCR product with the plasmid backbone 1.5 Transform E.coli

Steps to use the PCR products

PCR Clean-up

• Use PCR Clean-up kit to extract the DNA. This should result in 50ul of final product

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 fast digest EcoRI
- 3ul of fast digest 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

Extract the DNA from the gel

• Cut the gel fragment containing the DNA and extract the DNA. The final volume of the DNA will be 50ul.
• Run 5ul of extracted DNA on the gel to make sure it is right
• Measure the concentration of the DNA

Ligate the PCR product with the plasmid backbone

• 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 (PCR product)
- 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

• 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