Boston UniversityStatus

From 2007.igem.org

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==What We've Accomplished==
==What We've Accomplished==
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===Primer Design===
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[[Boston_UniversityPlasmidChoice | Plasmid Choice]]
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SO1415
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[[Boston_UniversityRestrictionChoice | Restriction Enzyme Choice]]
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Gene sequence: '''overhang''' of the actual gene
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[[Boston_UniversityPrimerDesign | Primer Design]]
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This sequence was found on http://www.ncbi.nlm.nih.gov/
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[[Boston_UniversityCompTransf | Making Shewanella Competent and Transforming Plasmid]]
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Megablast was used to compare the designed primer against the s. oneidensis genome
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The length, gc content, melt temp etc info was found on www.idtdna.com
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The site used to find parameters of a well-designed primer: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
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SO_1415 gene sequence:
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5’
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'''GAATGAATAA'''
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ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA
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TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC
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TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT
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CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT
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TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT
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GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT
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ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG
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TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT
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TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG
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CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG
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ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG
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TCAGTGTGGTTAG
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'''GGCGTCCTGC'''
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3’
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Primer 1:
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'''TGA ATA A'''AT GAA ATG TCC TTC GGA CTC CCT G
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LENGTH:31
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GC CONTENT:41.9 %
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MELT TEMP:59.9 ºC
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MOLECULAR WEIGHT:9494.2 g/mole
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EXTINCTION COEFFICIENT:298500 L/(mole·cm)
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nmole/OD260:3.35
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µg/OD260:31.81
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Primer 2:
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'''GAC GCC''' CTA ACC ACA CTG ACG
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LENGTH:21
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GC CONTENT:61.9 %
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MELT TEMP:60.2 ºC
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MOLECULAR WEIGHT:6345.2 g/mole
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EXTINCTION COEFFICIENT:197000 L/(mole·cm)
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nmole/OD260:5.08
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µg/OD260:32.21
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SO4157
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5’
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'''AAGGAAAACC'''
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ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
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TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
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ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA
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CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG
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CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA
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AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC
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GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT
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ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
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AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
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'''TCCAATAAAC'''
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3’
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Primer 1:
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'''AAA ACC''' ATG TCC ACC ATG CTG C
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LENGTH:22
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GC CONTENT:50.0 %
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MELT TEMP:58.7 ºC
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MOLECULAR WEIGHT:6648.4 g/mole
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EXTINCTION COEFFICIENT:207700 L/(mole·cm)
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nmole/OD260:4.81
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µg/OD260:32.01
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Primer 2:
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'''ATT GGA''' TTA CTC CGG TAA GAG TGT ATT TAG GT
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LENGTH:32
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GC CONTENT:37.5 %
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MELT TEMP:58.6 ºC
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MOLECULAR WEIGHT:9924.5 g/mole
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EXTINCTION COEFFICIENT:320800 L/(mole·cm)
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nmole/OD260:3.12
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µg/OD260:30.94
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hlyU
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'''ATGAAAACCA'''
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TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
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ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
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CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA
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AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
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ATCAATTCAATATTCAT
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'''TACGCACTTT'''
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Primer 1:
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'''ATG AAA ACC A'''TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG
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LENGTH:41
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GC CONTENT:22.0 %
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MELT TEMP:56.6 ºC
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MOLECULAR WEIGHT:12655.3 g/mole
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EXTINCTION COEFFICIENT:432600 L/(mole·cm)
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nmole/OD260:2.31
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µg/OD260:29.25
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Primer 2:
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'''GTG CGT A'''AT GAA TAT TGA ATT GAT GCA GGA
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LENGTH:30
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GC CONTENT:36.7 %
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MELT TEMP:57.8 ºC
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MOLECULAR WEIGHT:9349.1 g/mole
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EXTINCTION COEFFICIENT:309700 L/(mole·cm)
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nmole/OD260:3.23
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µg/OD260:30.19
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== Week's (Ambitious) Goals ==
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Wednesday 5/30
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# Get all protocols
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#  Identify materials/prepare order
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#  Design Primers
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#  Learn about budget/POs
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Thursday 5/31
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# Do primer order
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# Start conjugation practice
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# Confirm restriction enzymes, ligases
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# Order confirmed/needed materials
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# Team Revew Meeting
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# Draft Thank-You Letters for our Sponsors
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Friday 6/1
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# Evaluate/continue conjugation, practice electroporation for E. coli
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# Revise proposal to include possibility of screening with alginate beads and fluorocytometer
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# Meeting with Tim:  Budgets/protocols, Pfizer/fundraising, iGEM registration, beads
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Week of 6/4:
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1. Evaluate the transformation that was done on Friday.
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2. Confirm the correct plasmid (pJQ200)
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3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.
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4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.
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5. Order primers.
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6. Practice regular (non-error prone) PCR with the primers to check that they work.
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7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.
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8. Conjugate this plasmid into Shewy.
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== Materials We Need ==
== Materials We Need ==
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Primers:  Need to Buy
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Error-Prone PCR:  From CAB(?)
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Error-Prone PCR:  Need to Buy
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Ligases:  Need to Buy
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Plasmids:  Need to Buy?
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Restriction Enzymes:  Need to Buy?
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Ligases:  Need to Buy?
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== Short-Term To-Do List ==
== Short-Term To-Do List ==
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EDIT THE WIIIIKIIII
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Lab Orientation: COMPLETED!
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*[[Lab Orientation Checklist]]
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Lab Safety Training: COMPLETED!
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Design of Primers:  COMPLETED!
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Ordering of Primers:  COMPLETED!
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Gathering of Protocols:  Not Completed (Chris, please send me the protocols when they are gathered)
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Ordering of Error-Prone PCR Materials:  Not completed
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Thank-You Letters sent to Pfizer:  Not Completed
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Thank-You Letters sent to BU ppl:  Not completed
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== Protocols ==
== Protocols ==
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==="Calcium Chloride/Heat Shock Plasmid Transformations===
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[[Boston_UniversityHeatShockProtocol | Calcium Chloride/Heat Shock Plasmid Transformations Protocol]]
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Reagents to be Supplied by the User
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[[Boston_UniversityFilterConjugationProtocol | Filter Cojugation Protocol]]
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LB plates with appropriate antibiotic concentration (for pET -25b(+), amipicillin at 50-100 microgram/mL) SOC media
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[[Boston_UniversityTOPO | pTrcHis TOPO TA Expression Kit Cloning Protocol]]
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1) Prepare one LB-Amp plate for each transformation, plus one plate for a negative (no plasmid) control. After storage at 2-8 degrees C, equilibrate to room temperature.
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[[Boston_UniversityNEBCutter| Using NEBCutter for checking specific restriction enzymes against a sequence]]
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2) Centrifuge the tubes containing plasmid DNA to collect contents at the bottom of the tube. Add 1 microliter DNA to a sterile 1.5 mL tube on ice. Have one tube on ice with no DNA.
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[[Boston_UniversityGel | Making an Electrophoresis Gel]]
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3) Add 50 microliters of competent cells (either freshly prepared, or frozen and thawed on ice). Avoid excessive pipetting, as cells are very fragile.
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[[Boston_UniversityZymo/SOB | Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
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]]
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4) Gently flick the tubes to mix, and place on ice for 20 minutes.
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5) Heat shock the cells for 45 seconds to 2 minutes in a water bath at exactly 42 degrees C. Do not shake.
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6) Immediately return the tubes to ice for 2-10 minutes.
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7) Add 950 microliters of room temperature SOC media to the tubes.
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8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).
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9) Plate 100 microliters of each transformation culture onto antibiotic plates.
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10) Incubate the plates overnight (16-24 hours) at 37 degrees C.
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See Making Heat Shock Competent Cells for more information. "
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Negative Control:  No plasmid
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'''Trial 1:'''
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We followed this protocol from step 3 in order to practice our transformation and conjugation techniques. The competent cells given to us by Joshua were E.coli sm10; the GFP plasmids (pMS291/lacI) were supplied by Ilaria. But we performed the protocol with four samples of plasmid DNA+E.coli. Each sample contained 1 microliter of 63.5 ng/microliter GFP plasmid DNA; 50 microliters of the competent E.coli cells; and 950 microliters of SOC media. 100 microliters of each sample tube were plated onto one kanamycin plate and one blank (non-antibiotic), control plate.
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'''Trial 1 results:'''
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A lawn of bacteria on each plate. No GFP fluorescence under UV illumination.
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'''Trial 2:'''
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Used E.coli sm10 cells, GFP plasmid pMS291 with lacZ promoter (stimulated by IPTG). 
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Sample 1 contained 50 microliter sm10 + 1 microliter mPS291. 
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Sample 2 contained 50 microliter sm10 + 1 microliter water. 
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Sample 3 contained 50 microliter E.coli Dh5-alpha + 1 microliter water. 
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Samples 2 and 3 served as negative controls (should show no colonies in the presence of kanamycin). 
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5 microliters of each of these samples were plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG]. 
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50 microliters of each of these samples was also plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].
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'''Trial 2 results:'''
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Colony growth on all plates.  So the kanamycin is not killing any of the cells.  Perhaps the kan plates were made incorrectly.
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'''Trial 3:'''
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Repeated Trial 2 but with double volume of kanamycin and double volume of IPTG (to ensure stimulation of lacZ promoter an thus GFP expression).
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'''Trial 3 results:'''
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5 microliters sm10 + water in IPTG and kan: lawn of colonies
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50 microliters sm10 + water in IPTG and kan: less than 10 colonies
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5 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, '''fluorescent under UV illumination !'''
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50 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, no fluorescence
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5 microliters Dh5-alpha + water in IPTG and kan: more than 100 colonies
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5 microliters sm10 + water in kan: lawn of colonies
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5 microliters sm10 + pMS291 in kan: lawn of colonies
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5 microliters Dh5-alpha + water in kan: more than 100 isolated colonies
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E.coli successfully transformed with pMS291 (showed fluorescence).  These colonies will be grown out in SOC media  for further examination.  But kan still seems to be innefective.
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[update] The succesfully transformed E.coli that fluoresced were grown out in SOC media and then centrifuged to obtain a pellet of cells.  This pellet definitely fluoresced under UV confirming that the transformation was succesful.
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'''Trial 4:'''
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Two stocks were made: one with 20 microliters E.coli Dh5-alpha + pMS291 (lacZ), and the second with 20 microliters E.coli Dh5-alpha + water.  Each of these two stocks were plated onto 4 different plates.
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Plate 1 contained LB
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Plate 2 contained LB + kanamycin
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Plate 3 contained LB + kanamycin + IPTG
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Plate 4 contained LB + kanamycin which was dripped onto the LB
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'''Trial 4 Results:'''
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''20 microliters E.coli Dh5-alpha + pMS291 (lacZ)''
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Plate 1 containing LB: lawn of bacteria
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Plate 2 containing LB + kanamycin: more than 100 colonies
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Plate 3 containing LB + kanamycin + IPTG: more than 100 colonies and fluorescent!
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Plate 4 containing LB + kanamycin which was dripped onto the LB: more than 100 colonies
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''20 microliters E.coli Dh5-alpha + water''
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Plate 1 contained LB: no colonies
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Plate 2 contained LB + kanamycin: no colonies
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Plate 3 contained LB + kanamycin + IPTG: no colonies
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Plate 4 contained LB + kanamycin which was dripped onto the LB: no colonies
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Therefore, the kanamycin worked correctly. E.coli without pMS291 (without kanamycin resistance) all died. Transformation was also successful
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== Question and Answer ==
== Question and Answer ==
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== Relevant Publications and Links==
 
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http://www.shewybase.bu.edu
 
[https://2007.igem.org/Boston_University Back]
[https://2007.igem.org/Boston_University Back]

Latest revision as of 16:43, 6 July 2007

Back

Contents

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Making Shewanella Competent and Transforming Plasmid

Materials We Need

Error-Prone PCR: From CAB(?)

Ligases: Need to Buy

Short-Term To-Do List

EDIT THE WIIIIKIIII

Protocols

Calcium Chloride/Heat Shock Plasmid Transformations Protocol

Filter Cojugation Protocol

pTrcHis TOPO TA Expression Kit Cloning Protocol

Using NEBCutter for checking specific restriction enzymes against a sequence

Making an Electrophoresis Gel

Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB

Question and Answer

Back

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