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Revision as of 20:39, 31 May 2007 (view source) Dbellin (Talk | contribs) (→Primer Design) ← Older edit		Revision as of 20:41, 31 May 2007 (view source) Dbellin (Talk | contribs) (→Primer Design) Newer edit →
Line 4:		Line 4:
	===Primer Design===		===Primer Design===
		+
	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/		This sequence was found on http://www.ncbi.nlm.nih.gov/
Line 35:		Line 36:
	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 %
		+
	MELT TEMP:59.9 ºC		MELT TEMP:59.9 ºC
		+
	MOLECULAR WEIGHT:9494.2 g/mole		MOLECULAR WEIGHT:9494.2 g/mole
		+
	EXTINCTION COEFFICIENT:298500 L/(mole·cm)		EXTINCTION COEFFICIENT:298500 L/(mole·cm)
		+
	nmole/OD260:3.35		nmole/OD260:3.35
		+
	µg/OD260:31.81		µg/OD260:31.81
	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 %
		+
	MELT TEMP:60.2 ºC		MELT TEMP:60.2 ºC
		+
	MOLECULAR WEIGHT:6345.2 g/mole		MOLECULAR WEIGHT:6345.2 g/mole
		+
	EXTINCTION COEFFICIENT:197000 L/(mole·cm)		EXTINCTION COEFFICIENT:197000 L/(mole·cm)
		+
	nmole/OD260:5.08		nmole/OD260:5.08
		+
	µg/OD260:32.21		µg/OD260:32.21
Line 73:		Line 90:
	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 %
		+
	MELT TEMP:58.7 ºC		MELT TEMP:58.7 ºC
		+
	MOLECULAR WEIGHT:6648.4 g/mole		MOLECULAR WEIGHT:6648.4 g/mole
		+
	EXTINCTION COEFFICIENT:207700 L/(mole·cm)		EXTINCTION COEFFICIENT:207700 L/(mole·cm)
		+
	nmole/OD260:4.81		nmole/OD260:4.81
		+
	µg/OD260:32.01		µg/OD260:32.01
	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 %
		+
	MELT TEMP:58.6 ºC		MELT TEMP:58.6 ºC
		+
	MOLECULAR WEIGHT:9924.5 g/mole		MOLECULAR WEIGHT:9924.5 g/mole
		+
	EXTINCTION COEFFICIENT:320800 L/(mole·cm)		EXTINCTION COEFFICIENT:320800 L/(mole·cm)
		+
	nmole/OD260:3.12		nmole/OD260:3.12
		+
	µg/OD260:30.94		µg/OD260:30.94
		+
	hlyU		hlyU
Line 104:		Line 137:
	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 %
		+
	MELT TEMP:56.6 ºC		MELT TEMP:56.6 ºC
		+
	MOLECULAR WEIGHT:12655.3 g/mole		MOLECULAR WEIGHT:12655.3 g/mole
		+
	EXTINCTION COEFFICIENT:432600 L/(mole·cm)		EXTINCTION COEFFICIENT:432600 L/(mole·cm)
		+
	nmole/OD260:2.31		nmole/OD260:2.31
		+
	µg/OD260:29.25		µg/OD260:29.25
	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 %
		+
	MELT TEMP:57.8 ºC		MELT TEMP:57.8 ºC
		+
	MOLECULAR WEIGHT:9349.1 g/mole		MOLECULAR WEIGHT:9349.1 g/mole
		+
	EXTINCTION COEFFICIENT:309700 L/(mole·cm)		EXTINCTION COEFFICIENT:309700 L/(mole·cm)
		+
	nmole/OD260:3.23		nmole/OD260:3.23
		+
	µg/OD260:30.19		µg/OD260:30.19

---

Revision as of 20:41, 31 May 2007

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Contents 1 What We've Accomplished 1.1 Primer Design 2 Week's (Ambitious) Goals 3 Materials We Need 4 Short-Term To-Do List 5 Relevant Publications and Links

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 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 Orientation Checklist

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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