BU06:Team C
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| - | |<font size="4"> '''Team Goal: Add Restriction Sites to Biobrick Ends''' </font> | + | |<font size="4" font face="century gothic"> '''Team Goal: Add Restriction Sites to Biobrick Ends''' </font> |
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| - | back to the [[:Category: Boston University 2006|BU iGEM main page]] | + | <font face="century gothic"> back to the [[:Category: Boston University 2006|BU iGEM main page]] |
[[Category: Boston University 2006| Team C]] | [[Category: Boston University 2006| Team C]] | ||
| - | == Members == | + | == '''Members''' == |
{| width="400" | {| width="400" | ||
|- style="background:#E1E1E1;" | |- style="background:#E1E1E1;" | ||
| - | | '''Melissa Kinney''' | + | | <font face="century gothic">'''Melissa Kinney''' |
| - | | [mailto:mkinney@bu.edu mkinney@bu.edu] | + | | <font face="century gothic">[mailto:mkinney@bu.edu mkinney@bu.edu] |
|- | |- | ||
| - | | '''Felix Liu''' | + | | <font face="century gothic">'''Felix Liu''' |
| - | | [mailto:fliu@bu.edu fliu@bu.edu] | + | | <font face="century gothic">[mailto:fliu@bu.edu fliu@bu.edu] |
|- style="background:#E1E1E1;" | |- style="background:#E1E1E1;" | ||
| - | | '''Camilo Garcia''' | + | | <font face="century gothic">'''Camilo Garcia''' |
| - | | [mailto:camcom1p@bu.edu camcom1p@bu.edu] | + | | <font face="century gothic">[mailto:camcom1p@bu.edu camcom1p@bu.edu] |
|- | |- | ||
| - | | '''Billy Andre''' | + | | <font face="century gothic">'''Billy Andre''' |
| - | | [mailto:bandre@bu.edu bandre@bu.edu] | + | | <font face="century gothic">[mailto:bandre@bu.edu bandre@bu.edu] |
|} | |} | ||
| - | == Meeting Schedule == | + | == '''Meeting Schedule''' == |
Wednesday July 12th, 3pm Ingalls | Wednesday July 12th, 3pm Ingalls | ||
* Goal: Present info on how to isolate from plasmid, how to PCR and add ends, and protocol/primer design | * Goal: Present info on how to isolate from plasmid, how to PCR and add ends, and protocol/primer design | ||
** I posted a list of links to websites with protocols which should be helpful in designing a plan for our project. This week everyone should look through the websites and become familiar with some of the techniques. Try to create a rough outline of how to approach the experiment and what steps need to be taken from start to end. We have the [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=155405 sequence] for our luxCDABE operon, and we have identified AgeI as the restriction site that we will use in doing the PCR reaction. If you copy the sequence into NEB Cutter (link below) you can see where all of the restrition sites are, including AgeI. Start to think about how we would design primers and maybe even give it a shot. Feel free to post anything you find or any questions you encounter on the wiki so that we can use it as a forum for ideas and progress. You can also [mailto:mkinney@bu.edu e-mail] any questions or ideas to me and i'll put it up on the wiki. We'll meet to go over everything that we have found out this week and then present our progress at the general meeting. | ** I posted a list of links to websites with protocols which should be helpful in designing a plan for our project. This week everyone should look through the websites and become familiar with some of the techniques. Try to create a rough outline of how to approach the experiment and what steps need to be taken from start to end. We have the [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=155405 sequence] for our luxCDABE operon, and we have identified AgeI as the restriction site that we will use in doing the PCR reaction. If you copy the sequence into NEB Cutter (link below) you can see where all of the restrition sites are, including AgeI. Start to think about how we would design primers and maybe even give it a shot. Feel free to post anything you find or any questions you encounter on the wiki so that we can use it as a forum for ideas and progress. You can also [mailto:mkinney@bu.edu e-mail] any questions or ideas to me and i'll put it up on the wiki. We'll meet to go over everything that we have found out this week and then present our progress at the general meeting. | ||
| - | == Current Progress == | + | == '''Current Progress''' == |
* '''Isolation of luxCDABE''':The luxCDABE operon has been designed such that it can be removed from the pUC19 plasmid by EcoRI sites that are at the very beginning and ends. This makes it easy to isolate the operon from pUC19 in order to add the restriction sites to the ends. | * '''Isolation of luxCDABE''':The luxCDABE operon has been designed such that it can be removed from the pUC19 plasmid by EcoRI sites that are at the very beginning and ends. This makes it easy to isolate the operon from pUC19 in order to add the restriction sites to the ends. | ||
*'''Adding Ends''': PCR is one way to add restriction sites onto the ends of a sequence. Primers can be designed which will anneal to the gene, yet have an overhang which includes the restriction site(s). The problem arises in the length of our operon. At nearly 7000bp, the operon is too long for PCR to amplify all of it (standard PCR is only efficient up to about 5000bp according to our PCR master mix). Therefore, the PCR reaction must be preformed in different parts, cut with a restriction enzyme in the middle and ligated into one piece. We will use the AgeI restriction site in the middle where two PCR reactions will overlap. | *'''Adding Ends''': PCR is one way to add restriction sites onto the ends of a sequence. Primers can be designed which will anneal to the gene, yet have an overhang which includes the restriction site(s). The problem arises in the length of our operon. At nearly 7000bp, the operon is too long for PCR to amplify all of it (standard PCR is only efficient up to about 5000bp according to our PCR master mix). Therefore, the PCR reaction must be preformed in different parts, cut with a restriction enzyme in the middle and ligated into one piece. We will use the AgeI restriction site in the middle where two PCR reactions will overlap. | ||
| - | ===Questions=== | + | ==='''Questions'''=== |
* How can we test if the restriction sites have been added successfully? | * How can we test if the restriction sites have been added successfully? | ||
| - | == References == | + | == '''References''' == |
| - | === Techniques === | + | === '''Techniques''' === |
*[http://www.openwetware.org/wiki/Designing_primers Designing Primers] | *[http://www.openwetware.org/wiki/Designing_primers Designing Primers] | ||
*[http://www.mcb.uct.ac.za/pcroptim.htm More Info on Primer Design] | *[http://www.mcb.uct.ac.za/pcroptim.htm More Info on Primer Design] | ||
| - | === Protocols === | + | === '''Protocols''' === |
*[http://www1.qiagen.com/literature/BenchGuide/pdf/1017778_BenchGuide.pdf Qigaen Bench Guide] - Very good general overview of protocols | *[http://www1.qiagen.com/literature/BenchGuide/pdf/1017778_BenchGuide.pdf Qigaen Bench Guide] - Very good general overview of protocols | ||
*[http://www.openwetware.org/wiki/Agarose_gel_electrophoresis Agarose Gel Electrophoresis] | *[http://www.openwetware.org/wiki/Agarose_gel_electrophoresis Agarose Gel Electrophoresis] | ||
| Line 50: | Line 50: | ||
*[http://www.openwetware.org/wiki/Restriction_digest Restriction Digest] | *[http://www.openwetware.org/wiki/Restriction_digest Restriction Digest] | ||
| - | === Tools === | + | === '''Tools''' === |
*[http://tools.neb.com/NEBcutter2/index.php NEB Cutter] | *[http://tools.neb.com/NEBcutter2/index.php NEB Cutter] | ||
*[http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ IDT Oligo Analyzer] | *[http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ IDT Oligo Analyzer] | ||
Revision as of 16:57, 6 July 2006
|
| Schedule | Research | Fundraising | Resources | Members | Contact |
|---|
| Team Goal: Add Restriction Sites to Biobrick Ends |
back to the BU iGEM main page
Contents |
Members
| Melissa Kinney | mkinney@bu.edu |
| Felix Liu | fliu@bu.edu |
| Camilo Garcia | camcom1p@bu.edu |
| Billy Andre | bandre@bu.edu |
Meeting Schedule
Wednesday July 12th, 3pm Ingalls
- Goal: Present info on how to isolate from plasmid, how to PCR and add ends, and protocol/primer design
- I posted a list of links to websites with protocols which should be helpful in designing a plan for our project. This week everyone should look through the websites and become familiar with some of the techniques. Try to create a rough outline of how to approach the experiment and what steps need to be taken from start to end. We have the sequence for our luxCDABE operon, and we have identified AgeI as the restriction site that we will use in doing the PCR reaction. If you copy the sequence into NEB Cutter (link below) you can see where all of the restrition sites are, including AgeI. Start to think about how we would design primers and maybe even give it a shot. Feel free to post anything you find or any questions you encounter on the wiki so that we can use it as a forum for ideas and progress. You can also e-mail any questions or ideas to me and i'll put it up on the wiki. We'll meet to go over everything that we have found out this week and then present our progress at the general meeting.
Current Progress
- Isolation of luxCDABE:The luxCDABE operon has been designed such that it can be removed from the pUC19 plasmid by EcoRI sites that are at the very beginning and ends. This makes it easy to isolate the operon from pUC19 in order to add the restriction sites to the ends.
- Adding Ends: PCR is one way to add restriction sites onto the ends of a sequence. Primers can be designed which will anneal to the gene, yet have an overhang which includes the restriction site(s). The problem arises in the length of our operon. At nearly 7000bp, the operon is too long for PCR to amplify all of it (standard PCR is only efficient up to about 5000bp according to our PCR master mix). Therefore, the PCR reaction must be preformed in different parts, cut with a restriction enzyme in the middle and ligated into one piece. We will use the AgeI restriction site in the middle where two PCR reactions will overlap.
Questions
- How can we test if the restriction sites have been added successfully?
References
Techniques
Protocols
- Qigaen Bench Guide - Very good general overview of protocols
- Agarose Gel Electrophoresis
- DNA Ligation
- Miniprep
- QIAquick Spin Handbook - For PCR purification and gel extraction
- Restriction Digest
Tools
