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Revision as of 01:47, 1 June 2007 (view source) Dbellin (Talk | contribs) (→Primer Design) ← Older edit		Revision as of 16:33, 1 June 2007 (view source) Dbellin (Talk | contribs) (→Relevant Publications and Links) Newer edit →
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	Thank-You Letters sent to BU ppl:  Not completed		Thank-You Letters sent to BU ppl:  Not completed
		+
		+	== Protocols ==
		+	==="Calcium Chloride/Heat Shock Plasmid Transformations===
		+
		+	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.
		+
		+	8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).
		+
		+	9) Plate 100 microliters of each transformation culture onto antibiotic plates.
		+
		+	10) Incubate the plates overnight (16-24 hours) at 37 degrees C.
		+
		+	See Making Heat Shock Competent Cells for more information. "
		+
		+	Negative Control:  No plasmid
		+	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==		== Relevant Publications and Links==

---

Revision as of 16:33, 1 June 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 Protocols 5.1 "Calcium Chloride/Heat Shock Plasmid Transformations 6 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: 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

"Calcium Chloride/Heat Shock Plasmid Transformations

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.

8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).

9) Plate 100 microliters of each transformation culture onto antibiotic plates.

10) Incubate the plates overnight (16-24 hours) at 37 degrees C.

See Making Heat Shock Competent Cells for more information. "

Negative Control: No plasmid 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

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