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

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Revision as of 15:22, 1 September 2009

Meetings Calendar

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
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 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 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

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

Newcastle iGEM Twitter
[http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
[http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]

@@ Line 7: / Line 7: @@
 *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)
-Although the total will be 50ul, some of it will be consumed for the gel and to measure the concentration. Make a total solution of 70ul using the followings. (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
@@ Line 14: / Line 14: @@
   - 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
+*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 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 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
-Mix everything well and incubate at 37C for an hour
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