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

From 2009.igem.org

(Difference between revisions)
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{{:Team:Newcastle/Left}}
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=Steps to use the PCR products=
 +
 +
==PCR Clean-up==
*Use PCR Clean-up kit to clear the PCR product
*Use PCR Clean-up kit to clear the PCR product
This should result in 50ul of final product
This should result in 50ul of final product
*Measure the concentration of the 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
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)
+
==Digest the PCR product==
-
**Prepare a final volume of 70ul using
+
*Check the restriction sites and replace EcoRI and SpeI as you need)
 +
*Prepare a final volume of 70ul using
  - 50ul of the PCR product
  - 50ul of the PCR product
  - 3ul of EcoRI
  - 3ul of EcoRI
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  - 7ul of 10X buffer
  - 7ul of 10X buffer
  - Use water to make the final volume 70ul
  - Use water to make the final volume 70ul
-
**Mix everything well and incubate at 37C for an hour
+
*Mix everything well and incubate at 37C for an hour
-
**Run the total 70ul on the gel using large wells
+
*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 it. The final volume will be 50ul.
*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
*Run 5ul of extracted DNA on the gel to make sure it is right
 +
 +
==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
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  - 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 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

Revision as of 15:27, 1 September 2009

Contents

Steps to use the PCR products

PCR Clean-up

  • 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

Extract the DNA from the gel

  • 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 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
- 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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