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	==Notebook==		==Notebook==
-	[[Image:epfl_plasmid1.jpg|thumb|Ligation Progress Diagram]]	+	[[Image:Epfl_plasmid1.jpg|thumb|Progress Plasmid 1]]
		+	[[Image:Epfl_plasmid2.jpg|thumb|Progress Plasmid 2]]
		+
	{|border="0" cellpadding="5" align="center"		{|border="0" cellpadding="5" align="center"

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Revision as of 10:09, 29 August 2008

Contents 1 Notebook 2 Protocols 2.1 SOC medium 2.2 TE 2.3 SOB: Super Original Broth 2.4 CCMB80 buffer 2.5 Antibiotics 2.6 Transformation 2.7 Electrocompetent cells preparation 2.8 Target DNA Protocol 2.9 2Step PCR Reaction 2.10 Clean Room Protocol (for PDMS) 2.11 PDMS 2 layer device fab 3 Cloning Procedure

Notebook

July M T W T F S S   [http://2008.igem.org/EPF-Lausanne/1_July_2008 1] [http://2008.igem.org/EPF-Lausanne/2_July_2008 2] [http://2008.igem.org/EPF-Lausanne/3_July_2008 3] [http://2008.igem.org/EPF-Lausanne/4_July_2008 4] [http://2008.igem.org/EPF-Lausanne/5_July_2008 5] [http://2008.igem.org/EPF-Lausanne/6_July_2008 6] [http://2008.igem.org/EPF-Lausanne/7_July_2008 7] [http://2008.igem.org/EPF-Lausanne/8_July_2008 8] [http://2008.igem.org/EPF-Lausanne/9_July_2008 9] [http://2008.igem.org/EPF-Lausanne/10_July_2008 10] [http://2008.igem.org/EPF-Lausanne/11_July_2008 11] [http://2008.igem.org/EPF-Lausanne/12_July_2008 12] [http://2008.igem.org/EPF-Lausanne/13_July_2008 13] [http://2008.igem.org/EPF-Lausanne/14_July_2008 14] [http://2008.igem.org/EPF-Lausanne/15_July_2008 15] [http://2008.igem.org/EPF-Lausanne/16_July_2008 16] [http://2008.igem.org/EPF-Lausanne/17_July_2008 17] [http://2008.igem.org/EPF-Lausanne/18_July_2008 18] [http://2008.igem.org/EPF-Lausanne/19_July_2008 19] [http://2008.igem.org/EPF-Lausanne/20_July_2008 20] [http://2008.igem.org/EPF-Lausanne/21_July_2008 21] [http://2008.igem.org/EPF-Lausanne/22_July_2008 22] [http://2008.igem.org/EPF-Lausanne/23_July_2008 23] [http://2008.igem.org/EPF-Lausanne/24_July_2008 24] [http://2008.igem.org/EPF-Lausanne/25_July_2008 25] [http://2008.igem.org/EPF-Lausanne/26_July_2008 26] [http://2008.igem.org/EPF-Lausanne/27_July_2008 27] [http://2008.igem.org/EPF-Lausanne/28_July_2008 28] [http://2008.igem.org/EPF-Lausanne/29_July_2008 29] [http://2008.igem.org/EPF-Lausanne/30_July_2008 30] [http://2008.igem.org/EPF-Lausanne/31_July_2008 31]	August M T W T F S S         [http://2008.igem.org/EPF-Lausanne/1_August_2008 1] [http://2008.igem.org/EPF-Lausanne/2_August_2008 2] [http://2008.igem.org/EPF-Lausanne/3_August_2008 3] [http://2008.igem.org/EPF-Lausanne/4_August_2008 4] [http://2008.igem.org/EPF-Lausanne/5_August_2008 5] [http://2008.igem.org/EPF-Lausanne/6_August_2008 6] [http://2008.igem.org/EPF-Lausanne/7_August_2008 7] [http://2008.igem.org/EPF-Lausanne/8_August_2008 8] [http://2008.igem.org/EPF-Lausanne/9_August_2008 9] [http://2008.igem.org/EPF-Lausanne/10_August_2008 10] [http://2008.igem.org/EPF-Lausanne/11_August_2008 11] [http://2008.igem.org/EPF-Lausanne/12_August_2008 12] [http://2008.igem.org/EPF-Lausanne/13_August_2008 13] [http://2008.igem.org/EPF-Lausanne/14_August_2008 14] [http://2008.igem.org/EPF-Lausanne/15_August_2008 15] [http://2008.igem.org/EPF-Lausanne/16_August_2008 16] [http://2008.igem.org/EPF-Lausanne/17_August_2008 17] [http://2008.igem.org/EPF-Lausanne/18_August_2008 18] [http://2008.igem.org/EPF-Lausanne/19_August_2008 19] [http://2008.igem.org/EPF-Lausanne/20_August_2008 20] [http://2008.igem.org/EPF-Lausanne/21_August_2008 21] [http://2008.igem.org/EPF-Lausanne/22_August_2008 22] [http://2008.igem.org/EPF-Lausanne/23_August_2008 23] [http://2008.igem.org/EPF-Lausanne/24_August_2008 24] [http://2008.igem.org/EPF-Lausanne/25_August_2008 25] [http://2008.igem.org/EPF-Lausanne/26_August_2008 26] [http://2008.igem.org/EPF-Lausanne/27_August_2008 27] [http://2008.igem.org/EPF-Lausanne/28_August_2008 28] [http://2008.igem.org/EPF-Lausanne/29_August_2008 29] [http://2008.igem.org/EPF-Lausanne/30_August_2008 30] [http://2008.igem.org/EPF-Lausanne/31_August_2008 31]	September M T W T F S S [http://2008.igem.org/EPF-Lausanne/1_September_2008 1] [http://2008.igem.org/EPF-Lausanne/2_September_2008 2] [http://2008.igem.org/EPF-Lausanne/3_September_2008 3] [http://2008.igem.org/EPF-Lausanne/4_September_2008 4] [http://2008.igem.org/EPF-Lausanne/5_September_2008 5] [http://2008.igem.org/EPF-Lausanne/6_September_2008 6] [http://2008.igem.org/EPF-Lausanne/7_September_2008 7] [http://2008.igem.org/EPF-Lausanne/8_September_2008 8] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/9_September_2008&action=edit 9] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/10_September_2008&action=edit 10] [http://2008.igem.org/EPF-Lausanne/11_September_2008 11] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/12_September_2008&action=edit 12] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/14_September_2008&action=edit 14] [http://2008.igem.org/EPF-Lausanne/15_September_2008 15] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/16_September_2008&action=edit 16] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/17_September_2008&action=edit 17] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/18_September_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/19_September_2008&action=edit 19] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/21_September_2008&action=edit 21] [http://2008.igem.org/EPF-Lausanne/22_September_2008 22] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/23_September_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/24_September_2008&action=edit 24] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/25_September_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/26_September_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/28_September_2008&action=edit 28] [http://2008.igem.org/EPF-Lausanne/29_September_2008 29] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/30_September_2008&action=edit 30]	October M T W T F S S     [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/1_October_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/2_October_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/3_October_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/5_October_2008&action=edit 5] [http://2008.igem.org/EPF-Lausanne/6_October_2008 6] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/7_October_2008&action=edit 7] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/8_October_2008&action=edit 8] [http://2008.igem.org/EPF-Lausanne/9_October_2008 9] [http://2008.igem.org/EPF-Lausanne/10_October_2008 10] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/12_October_2008&action=edit 12] [http://2008.igem.org/EPF-Lausanne/13_October_2008 13] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/14_October_2008&action=edit 14] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/15_October_2008&action=edit 15] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/16_October_2008&action=edit 16] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/17_October_2008&action=edit 17] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/19_October_2008&action=edit 19] [http://2008.igem.org/EPF-Lausanne/20_October_2008 20] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/21_October_2008&action=edit 21] [http://2008.igem.org/EPF-Lausanne/22_October_2008 22] [http://2008.igem.org/EPF-Lausanne/23_October_2008 23] [http://2008.igem.org/EPF-Lausanne/24_October_2008 24] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/26_October_2008&action=edit 26] [http://2008.igem.org/EPF-Lausanne/27_October_2008 27] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/28_October_2008&action=edit 28] [http://2008.igem.org/EPF-Lausanne/29_October_2008 29] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/31_October_2008&action=edit 31]

Protocols

Reference : Most of these protocols come from http://openwetware.org

SOC medium

Super Original Catabolite Repressor

Bactotryptone                         20g.
Bacto-yeast extract                   5g.
NaCl                                  0.5g.
1M KCl                                2.5ml
ddH2O                                 to 1000 ml
Total Volume                          1000ml

Adjust pH to 7, with 10N NaOH. Autoclave to sterilize Add 20 ml of 1M glucose before use.

TE

10xTE for 1 liter from stock solutions 10 ml 1M Tris-HCl pH 8.0 2 ml 0.5M EDTA pH 8.0 988 ml ddH2O

→ 10xTE is 10 mM Tris-HCl and 1 mM EDTA

   * For the Tris-HCl use Tris base and adjust to desired pH using HCl.

SOB: Super Original Broth

Used in growing bacteria for preparing chemically competent cells Ingredients

   * 0.5% (w/v) yeast extract
   * 2% (w/v) tryptone
   * 10 mM NaCl
   * 2.5 mM KCl
   * 20 mM MgSO4

Per liter:

   * 5 g yeast extract
   * 20 g tryptone
   * 0.584 g NaCl
   * 0.186 g KCl
   * 2.4 g MgSO4

!!! Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

CCMB80 buffer

For 1L

   * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
   * 80 mM CaCl2.2H2O (11.8 g/L)
   * 20 mM MnCl2.4H2O (4.0 g/L)
   * 10 mM MgCl2.6H2O (2.0 g/L)
   * 10% glycerol (100 ml/L)
   * adjust pH DOWN to 6.4 with 0.1N HCl if necessary
         o adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
   * sterile filter and store at 4°C
   * slight dark precipitate appears not to affect its function

Antibiotics

For each antibiotic, solutions of 5 ml have been done and stored at -20°C (09.07.08)

Transformation

We used DH5-alpha cells from Bart's lab stock. We therefore followed the InVitrogen protocol.

Electrocompetent cells preparation

From segall-lab protocol

Target DNA Protocol

Materials:

5’Comp Cy5 primer

• 5’Cy5-GTC ATA CCG CCG GA
• order from IDT at 1umole, HPLC purified
• suspend to 500uM

Library primers:

• See spreadsheet for details
• 5’ Linker - BINDING SITE – 3’ Linker – TCCGGCGGTATGAC
• 5’ Linker generally: AAC
• Binding Site Generally a 12mer but can be longer
• 3’Linker generally: C
• order from IDT in a 96 well v-bottom plate suspended at 150uM in TE

Klenow Fragment 3’-5’ exo-

• Order from NEB (Cat #: M0212L)

dNTPs at 10mM each

• order from Roche (PCR Nucleotide Mix: 11581295001)

Method:

Synthesis reaction: 3uL dNTP 3uL Buffer 2 2uL Library primer 0.4uL 5’Comp Cy5 primer 1uL Klenow 3’-5’ exo- 20.6uL dH2O 30uL Final Volume

Prepare a master mix without the library primer, and load 28uL into a 96 well plate. Add 2uL of Library primer to the 96 well plate. Cycle as follows:

• 37°C for 1 hour
• 75°C for 20 mins
• ramp down to 30°C at 0.1°C/sec
• hold at 4°C

After the synthesis add 70uL of 0.5% BSA in dH2O to each well and transfer to a 384 well plate (flat bottom). Prepare dilutions as needed using 0.5% BSA dH2O.

To set up the 384 load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.

Transfer the column 1 of the klenow reaction (100uL total) to column 1 row A (A1) on the 384 well plate. Perform 5 dilutions of 70uL sample into the adjacent wells containing 30uL BSA dH2O. After the 5th dilution (in column 6) discard the remaining 70uL.

Transfer column 2 of the klenow plate into A7 and perform dilutions.

Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7, col7->B13, col8 -> B19

You should have 30uL of target DNA in each well of the 384 plate now. Add 30uL of 0.5% BSA dH2O to each well.

The samples are now ready to be spotted.

2Step PCR Reaction

Purpose: generate linear PCR templates for in vitro transcription/translation of cDNA clones using E.coli colonies as starting material. Should also be able to amplify genomic targets.

Template: E.coli colonies picked from agar plates Primer stock: 5’ + 3’ gene specific primers each at 500M Primers pair: 5’+3’ gene specific primers 1/10 in dH2O to 50M each Polymerase: HiFi Plus (Roche)

• suspend colony in 2.5uL lyse ‘n go buffer (Pierce)
• optionally 2.5uL of an overnight LB culture can also be used (adjust dH2O accordingly)
• heat to 95°C for 7 minutes then cool to 4°C
• add 1uL primer pair and 46.5 uL PCR Mix

PCR Mix: 1uL dNTP 10uL 5x Buffer + MgCl2 .5uL HiFi DNA polymerase 35uL dH2O 46.5uL Final Volume

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 55°C 1:00 4 72°C 2:00 5 Cycle 2-4 30x 6 72°C 7:00 7 4°C 

• run 1uL of product on gel to check for presence and length
• optionally: run a Qiagen PCR purification

2nd step of PCR

• 5’ext1 and 3’ext2 each at 50M
• Primer Mix: 5’ext1 + 3’ext2 primers diluted 1/200 in dH2O to 250nM each
• Template = previous PCR mix

1uL dNTP 10uL 5x Buffer + MgCl2 1uL Primer Mix 0.5uL template 0.5uL HiFi Plus DNAp 37uL dH2O 50uL Final Volume

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 55°C 1:00 4 72°C 2:00 5 Cycle 2-4 10x 6 72°C 7:00 7 4°C 

• 5’Final and 3’Final at 500M
• Final Primer Mix: 5’Final and 3’Final diluted 1/10 in dH2O
• add 1uL of Final Primer Mix

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 50°C 1:00 4 72°C 2:00 5 Cycle 2-4 25-30x 6 72°C 7:00 7 4°C 

• check 1uL product on a 1% agarose gel
• no further purification is necessary even though PCR purification is recommended, eluting in 50uL of elution buffer (EB) or pH adjusted dH2O

Clean Room Protocol (for PDMS)

Clean Room Protocol for preparing the molds for creating the PDMS devices

Coming soon

PDMS 2 layer device fab

Materials:

• flow and control layer molds
• Sylgard 184 part A and part B
• TMCS

Method:

• Place molds into a TMCS vapor chamber
• Control layer mixture: 30g Part A + 6g Part B
• Mix for 1 minute, degas for 2 minutes
• pour onto control layer mold and place mold in vacuum chamber
• Flow layer mixture: 30g Part A + 1.5g Part B
• Mix for 1 minute and degas for 2 minutes
• Spin coat onto flow layer at 2600-3000rpm for 30secs
• Remove control layer mold from vacuum chamber, making sure no bubbles are left on the surface.
• Place the control and flow layer in a 80C convection oven and incubate for 30 minutes
• Remove casts from oven, cut out control layer, punch holes, and align to flow layer
• Put aligned device back into 80C oven and incubate for at least 90 minutes.
• Remove devices from oven and punch flow layer holes

Cloning Procedure

Biobrick 1 (insert)	Biobrick 2 (vector)	Digestion	Ligation	Worked ?
F1610 (LuxI protein generator)	E1010 (RFP coding sequence)	30/7	31/7	No
I14033 (constitutive promoter)	I1466 (RhlR protein generator)	30/7	31/7	No
R0071 (RhlR regulated promoter)	B0034 (RBS)	30/7	31/7	Maybe
R0040 (TetR regulated promoter)	B0034 (RBS)	30/7	31/7	Maybe

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