Boston UniversityStatus

From 2007.igem.org

(Difference between revisions)
(→Primer Design)
(→Primer Design)
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SO1415
SO1415
-
· Gene sequence: ‘’’Overhang’’’ of the actual gene
+
Gene sequence: '''overhang'''of the actual gene
-
· This sequence was found on http://www.ncbi.nlm.nih.gov/
+
 
-
· Megablast was used to compare the designed primer against the s.  
+
This sequence was found on http://www.ncbi.nlm.nih.gov/
-
oneidensis genome
+
 
-
· the length, gc content, melt temp etc info was found on www.idtdna.com
+
Megablast was used to compare the designed primer against the s. oneidensis genome
-
· the site used to find parameters of a well-designed primer:
+
 
-
      http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
+
The length, gc content, melt temp etc info was found on www.idtdna.com
 +
 
 +
The site used to find parameters of a well-designed primer: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
SO_1415 gene sequence:
SO_1415 gene sequence:
5’
5’
-
‘’’GAATGAATAA’’’
+
'''GAATGAATAA'''
ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA
ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA
TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC
TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC
Line 33: Line 35:
Primer 1:
Primer 1:
-
‘’’TGA ATA A’’’AT GAA ATG TCC TTC GGA CTC CCT G
+
'''TGA ATA A'''AT GAA ATG TCC TTC GGA CTC CCT G
LENGTH:31
LENGTH:31
GC CONTENT:41.9 %
GC CONTENT:41.9 %
Line 43: Line 45:
Primer 2:
Primer 2:
-
‘’’GAC GCC’’’ CTA ACC ACA CTG ACG
+
'''GAC GCC''' CTA ACC ACA CTG ACG
LENGTH:21
LENGTH:21
GC CONTENT:61.9 %
GC CONTENT:61.9 %
Line 57: Line 59:
5’
5’
-
‘’’AAGGAAAACC’’’
+
'''AAGGAAAACC'''
ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
Line 67: Line 69:
ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
-
‘’’TCCAATAAAC’’’
+
'''TCCAATAAAC'''
3’
3’
Primer 1:
Primer 1:
-
‘’’AAA ACC’’’ ATG TCC ACC ATG CTG C
+
'''AAA ACC''' ATG TCC ACC ATG CTG C
LENGTH:22
LENGTH:22
GC CONTENT:50.0 %
GC CONTENT:50.0 %
Line 81: Line 83:
Primer 2:
Primer 2:
-
‘’’ATT GGA’’’ TTA CTC CGG TAA GAG TGT ATT TAG GT
+
'''ATT GGA''' TTA CTC CGG TAA GAG TGT ATT TAG GT
LENGTH:32
LENGTH:32
GC CONTENT:37.5 %
GC CONTENT:37.5 %
Line 89: Line 91:
nmole/OD260:3.12
nmole/OD260:3.12
µg/OD260:30.94
µg/OD260:30.94
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
hlyU
hlyU
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‘’’ATGAAAACCA’’’
+
'''ATGAAAACCA'''
TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
Line 110: Line 100:
AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
ATCAATTCAATATTCAT
ATCAATTCAATATTCAT
-
‘’’TACGCACTTT’’’
+
'''TACGCACTTT'''
Primer 1:
Primer 1:
-
‘’’ATG AAA ACC A’’’TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG
+
'''ATG AAA ACC A'''TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG
LENGTH:41
LENGTH:41
GC CONTENT:22.0 %
GC CONTENT:22.0 %
Line 123: Line 113:
Primer 2:
Primer 2:
-
‘’’GTG CGT A’’’AT GAA TAT TGA ATT GAT GCA GGA
+
'''GTG CGT A'''AT GAA TAT TGA ATT GAT GCA GGA
LENGTH:30
LENGTH:30
GC CONTENT:36.7 %
GC CONTENT:36.7 %

Revision as of 20:39, 31 May 2007

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Contents

What We've Accomplished

Primer Design

SO1415

Gene sequence: overhangof the actual gene

This sequence was found on http://www.ncbi.nlm.nih.gov/

Megablast was used to compare the designed primer against the s. oneidensis genome

The length, gc content, melt temp etc info was found on www.idtdna.com

The site used to find parameters of a well-designed primer: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

SO_1415 gene sequence: 5’ GAATGAATAA ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG TCAGTGTGGTTAG ‘’’GGCGTCCTGC’’’ 3’

Primer 1: TGA ATA AAT GAA ATG TCC TTC GGA CTC CCT G LENGTH:31 GC CONTENT:41.9 % MELT TEMP:59.9 ºC MOLECULAR WEIGHT:9494.2 g/mole EXTINCTION COEFFICIENT:298500 L/(mole·cm) nmole/OD260:3.35 µg/OD260:31.81

Primer 2: GAC GCC CTA ACC ACA CTG ACG LENGTH:21 GC CONTENT:61.9 % MELT TEMP:60.2 ºC MOLECULAR WEIGHT:6345.2 g/mole EXTINCTION COEFFICIENT:197000 L/(mole·cm) nmole/OD260:5.08 µg/OD260:32.21


SO4157

5’ AAGGAAAACC ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA TCCAATAAAC 3’

Primer 1: AAA ACC ATG TCC ACC ATG CTG C LENGTH:22 GC CONTENT:50.0 % MELT TEMP:58.7 ºC MOLECULAR WEIGHT:6648.4 g/mole EXTINCTION COEFFICIENT:207700 L/(mole·cm) nmole/OD260:4.81 µg/OD260:32.01

Primer 2: ATT GGA TTA CTC CGG TAA GAG TGT ATT TAG GT LENGTH:32 GC CONTENT:37.5 % MELT TEMP:58.6 ºC MOLECULAR WEIGHT:9924.5 g/mole EXTINCTION COEFFICIENT:320800 L/(mole·cm) nmole/OD260:3.12 µg/OD260:30.94

hlyU

ATGAAAACCA TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC ATCAATTCAATATTCAT TACGCACTTT

Primer 1: ATG AAA ACC ATT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG LENGTH:41 GC CONTENT:22.0 % MELT TEMP:56.6 ºC MOLECULAR WEIGHT:12655.3 g/mole EXTINCTION COEFFICIENT:432600 L/(mole·cm) nmole/OD260:2.31 µg/OD260:29.25

Primer 2: GTG CGT AAT GAA TAT TGA ATT GAT GCA GGA LENGTH:30 GC CONTENT:36.7 % MELT TEMP:57.8 ºC MOLECULAR WEIGHT:9349.1 g/mole EXTINCTION COEFFICIENT:309700 L/(mole·cm) nmole/OD260:3.23 µg/OD260:30.19

Week's (Ambitious) Goals

Wednesday 5/30

  1. Get all protocols
  2. Identify materials/prepare order
  3. Design Primers
  4. Learn about budget/POs

Thursday 5/31

  1. Do primer order
  2. Start conjugation practice
  3. Confirm restriction enzymes, ligases
  4. Order confirmed/needed materials
  5. Team Revew Meeting
  6. Draft Thank-You Letters for our Sponsors

Friday 6/1

  1. Evaluate/continue conjugation, practice electroporation for E. coli
  2. Revise proposal to include possibility of screening with alginate beads and fluorocytometer
  3. Meeting with Tim: Budgets/protocols, Pfizer/fundraising, iGEM registration, beads

Materials We Need

Primers: Need to Buy

Error-Prone PCR: Need to Buy

Plasmids: Need to Buy?

Restriction Enzymes: Need to Buy?

Ligases: Need to Buy?

Short-Term To-Do List

Lab Orientation: COMPLETED!

Lab Safety Training: Not Completed (Dave, Rahul, and Christian are scheduled for a training session on 5/30/07. Danny will be taking it next week)

Design of Primers: Not Completed (Dave, please send me info about the finished primers when ready)

Ordering of Primers: Not Completed

Gathering of Protocols: Not Completed (Chris, please send me the protocols when they are gathered)

Ordering of Error-Prone PCR Materials: Not completed

Thank-You Letters sent to Pfizer: COMPLETED!

Thank-You Letters sent to BU ppl: Not completed

Relevant Publications and Links

http://www.shewybase.bu.edu

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