Trouble Shooting Davidson Design - Revision history

Cadoyle at 17:14, 9 June 2011

2011-06-09T17:14:03Z

Cadoyle at 16:03, 9 June 2011

2011-06-09T16:03:26Z

Cadoyle at 15:59, 9 June 2011

2011-06-09T15:59:07Z

Cadoyle at 15:58, 9 June 2011

2011-06-09T15:58:00Z

Cadoyle: Created page with 'We ran into a problem with our design. We designed the half edges with BsaI sites in both the middle and the ends of the half edges. Therefore when we digest with BsaI and perfor…'

2011-06-09T15:56:25Z

Created page with 'We ran into a problem with our design. We designed the half edges with BsaI sites in both the middle and the ends of the half edges. Therefore when we digest with BsaI and perfor…'

New page

We ran into a problem with our design. We designed the half edges with BsaI sites in both the middle and the ends of the half edges. Therefore when we digest with BsaI and perform Golden Gate shuffling we will get complete edges that are incorrect and to long.

See General Primer to design to look at the problem in out design [[Media:ggsdiagram.pptx‎ ]]<br>

Possible Solution:

After using Golden Gate Shuffling to create our complete edges we will perform another round of PCR with original primers. This will create many different sizes of the correct sequence of complete edges. Then we can run a gel and gel purify for the correct complete edge based on the number of bp.

To Avoid this Problem in the Future:

We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.

Possible Enzymes Available To Create Sticky Ends Similar to BsaI: [[AlternativeRestrictionEnzymes.png ]]<br>

← Older revision		Revision as of 15:59, 9 June 2011
Line 11:		Line 11:
	We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.		We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.

−	Possible Enzymes Available To Create Sticky Ends Similar to BsaI: [[~~File~~:AlternativeRestrictionEnzymes.png ]]<br>	+	Possible Enzymes Available To Create Sticky Ends Similar to BsaI: [[Media:AlternativeRestrictionEnzymes.png ]]<br>

← Older revision		Revision as of 15:58, 9 June 2011
Line 11:		Line 11:
	We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.		We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.

−	Possible Enzymes Available To Create Sticky Ends Similar to BsaI: [[AlternativeRestrictionEnzymes.png ]]<br>	+	Possible Enzymes Available To Create Sticky Ends Similar to BsaI: [[File:AlternativeRestrictionEnzymes.png ]]<br>

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 See General Primer to design to look at the problem in out design [[Media:ggsdiagram.pptx‎  ]]<br>
 <hr>
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 After using Golden Gate Shuffling to create our complete edges we will perform another round of PCR with original primers. This will create many different sizes of the correct sequence of complete edges. Then we can run a gel and gel purify for the correct complete edge based on the number of bp.
 <hr>

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-We ran into a problem with our design. We designed the half edges with BsaI sites in both the middle and the ends of the half edges. Therefore when we digest with BsaI and perform Golden Gate shuffling we will get complete edges that are incorrect and to long.
+Problem with Design:
 See General Primer to design to look at the problem in out design [[Media:ggsdiagram.pptx‎  ]]<br>
 Possible Solution:
 After using Golden Gate Shuffling to create our complete edges we will perform another round of PCR with original primers. This will create many different sizes of the correct sequence of complete edges. Then we can run a gel and gel purify for the correct complete edge based on the number of bp.
 To Avoid this Problem in the Future: