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

From 2009.igem.org

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(→Steps to use the PCR products)
 
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==PCR Clean-up==
==PCR Clean-up==
-
*Use PCR Clean-up kit to clear the PCR product
+
*Use PCR Clean-up kit to extract the DNA. This should result in 50ul of final 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==
==Digest the PCR product==
*Check the restriction sites and replace EcoRI and SpeI as you need)
*Check the restriction sites and replace EcoRI and SpeI as you need)
*Prepare a final volume of 70ul using
*Prepare a final volume of 70ul using
  - 50ul of the PCR product
  - 50ul of the PCR product
-
  - 3ul of EcoRI
+
  - 3ul of fast digest EcoRI
-
  - 3ul of SpeI
+
  - 3ul of fast digest SpeI
  - 7ul of 10X buffer
  - 7ul of 10X buffer
  - Use water to make the final volume 70ul
  - Use water to make the final volume 70ul
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==Extract the DNA from the gel==
==Extract the DNA from the gel==
-
*Cut the gel fragment containing the DNA and extract it. The final volume will be 50ul.
+
*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
*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 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)
*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
+
*Prepare a final volume of 30ul using
  - 3ul of ligation buffer(it is in the freezer)
  - 3ul of ligation buffer(it is in the freezer)
  - 1ul of ligase
  - 1ul of ligase
  - x (To be decided) ul of the backbone
  - x (To be decided) ul of the backbone
-
  - y (To be decided) ul of the insert
+
  - y (To be decided) ul of the insert (PCR product)
  - Water totalling to 30ul
  - 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
+
*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==
*Transform E.coli with the plasmid containing the insert
*Transform E.coli with the plasmid containing the insert
**Use the total 30ul for the transformation
**Use the total 30ul for the transformation
-
**For + control use the plasmid without any insert
+
**For (+) control, use the plasmid without any insert
-
**For - control use water to transform
+
**For (-) control, use water to transform

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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