Team:Newcastle/Project/Labwork/OurProtocols/PCR Products

=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
- 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
 * Check the restriction sites and replace EcoRI and SpeI as you need)
 * Prepare a final volume of 70ul using
 * 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
- 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
 * 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
 * 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