Boston UniversityStatus

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What We've Accomplished

Primer Design

SO1415

· 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. 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 A’’’AT 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 A’’’TT 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 A’’’AT 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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