Boston UniversityStatus
From 2007.igem.org
Contents |
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
- Get all protocols
- Identify materials/prepare order
- Design Primers
- Learn about budget/POs
Thursday 5/31
- Do primer order
- Start conjugation practice
- Confirm restriction enzymes, ligases
- Order confirmed/needed materials
- Team Revew Meeting
- Draft Thank-You Letters for our Sponsors
Friday 6/1
- Evaluate/continue conjugation, practice electroporation for E. coli
- Revise proposal to include possibility of screening with alginate beads and fluorocytometer
- 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
