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

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(New page: *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 ...)
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*Use PCR Clean-up kit to clear the PCR product
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This should result in 50ul of final product
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*Measure the concentration of the product
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This step will will be useful to determine how much of the product to use for the ligation
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*Digest the PCR product
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=Steps to use the PCR products=
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Although the total will be 50ul, some of it will be consumed for the gel and to measure the concentration
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==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==
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*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
 +
 
 +
 
 +
 
 +
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Latest revision as of 17:10, 2 September 2009

Contents

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




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