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	==What We've Accomplished==		==What We've Accomplished==
-	===Primer Design===	+	[[Boston_UniversityPlasmidChoice | Plasmid Choice]]
-	SO1415	+	[[Boston_UniversityRestrictionChoice | Restriction Enzyme Choice]]
-	Gene sequence: '''overhang''' of the actual gene	+	[[Boston_UniversityPrimerDesign | Primer Design]]
-	This sequence was found on http://www.ncbi.nlm.nih.gov/	+	[[Boston_UniversityCompTransf | Making Shewanella Competent and Transforming Plasmid]]
-		+
-	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 ==		== Materials We Need ==
-	Primers:  Need to Buy	+	Error-Prone PCR:  From CAB(?)
-	Error-Prone PCR:  Need to Buy	+	Ligases:  Need to Buy
-		+
-	Plasmids:  Need to Buy?	+
-		+
-	Restriction Enzymes:  Need to Buy?	+
-		+
-	Ligases:  Need to Buy?	+
	== Short-Term To-Do List ==		== Short-Term To-Do List ==
-		+	EDIT THE WIIIIKIIII
-	Lab Orientation: COMPLETED!	+
-	*[[Lab Orientation Checklist]]	+
-		+
-	Lab Safety Training: COMPLETED!	+
-		+
-	Design of Primers:  COMPLETED!	+
-		+
-	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:  Not Completed	+
-		+
-	Thank-You Letters sent to BU ppl:  Not completed	+
	== Protocols ==		== Protocols ==
-	==="Calcium Chloride/Heat Shock Plasmid Transformations===	+	[[Boston_UniversityHeatShockProtocol | Calcium Chloride/Heat Shock Plasmid Transformations Protocol]]
-		+
-	Reagents to be Supplied by the User	+
-		+
-	LB plates with appropriate antibiotic concentration (for pET -25b(+), amipicillin at 50-100 microgram/mL) SOC media	+
-		+
-	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.	+
-		+
-	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.	+
-		+
-	3) Add 50 microliters of competent cells (either freshly prepared, or frozen and thawed on ice). Avoid excessive pipetting, as cells are very fragile.	+
-		+
-	4) Gently flick the tubes to mix, and place on ice for 20 minutes.	+
-		+
-	5) Heat shock the cells for 45 seconds to 2 minutes in a water bath at exactly 42 degrees C. Do not shake.	+
-		+
-	6) Immediately return the tubes to ice for 2-10 minutes.	+
-	7) Add 950 microliters of room temperature SOC media to the tubes.	+	[[Boston_UniversityFilterConjugationProtocol | Filter Cojugation Protocol]]
-	8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).	+	[[Boston_UniversityTOPO | pTrcHis TOPO TA Expression Kit Cloning Protocol]]
-	9) Plate 100 microliters of each transformation culture onto antibiotic plates.	+	[[Boston_UniversityNEBCutter| Using NEBCutter for checking specific restriction enzymes against a sequence]]
-	10) Incubate the plates overnight (16-24 hours) at 37 degrees C.	+	[[Boston_UniversityGel | Making an Electrophoresis Gel]]
-	See Making Heat Shock Competent Cells for more information. "	+	[[Boston_UniversityZymo/SOB | Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
		+	]]
-	Negative Control:  No plasmid	+	== Question and Answer ==
-	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.	+
-	== Relevant Publications and Links==
-	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

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Contents 1 What We've Accomplished 2 Materials We Need 3 Short-Term To-Do List 4 Protocols 5 Question and Answer

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