Team:EPF-Lausanne/Notebook

From 2008.igem.org

(Difference between revisions)
(→Protocols)
Line 99: Line 99:
'''Electrocompetent cells preparation'''  
'''Electrocompetent cells preparation'''  
From segall-lab protocol
From segall-lab 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:
 +
3L dNTP
 +
3L Buffer 2
 +
2L Library primer
 +
0.4L 5’Comp Cy5 primer
 +
1L Klenow 3’-5’ exo-
 +
20.6L dH2O
 +
30L Final Volume
 +
 +
 +
 +
 +
 +
 +
 +
 +
Prepare a master mix without the library primer, and load 28L into a 96 well plate. Add 2L 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 70L 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.
 +
 +
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 2.5L lyse ‘n go buffer (Pierce)
 +
o optionally 2.5L 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 1L primer pair and 46.5 L PCR Mix
 +
 +
PCR Mix:
 +
1L dNTP
 +
10L 5x Buffer + MgCl2
 +
.5L HiFi DNA polymerase
 +
35L dH2O
 +
46.5L 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 1L 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
 +
 +
1L dNTP
 +
10L 5x Buffer + MgCl2
 +
1L Primer Mix
 +
0.5L template
 +
0.5L HiFi Plus DNAp
 +
37L dH2O
 +
50L 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 1L 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 1L product on a 1% agarose gel
 +
• no further purification is necessary even though PCR purification is recommended, eluting in 50L of elution buffer (EB) or pH adjusted dH2O
 +
 +
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

Revision as of 11:55, 30 July 2008


Home The Team The Project Parts Submitted to the Registry Modeling Notebook

Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Find more information on how to use the calendar feature by going to the general calendar page.


April
MTWTFSS
  [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/1_April_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/2_April_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/3_April_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/4_April_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/5_April_2008&action=edit 5] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/6_April_2008&action=edit 6]
[http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/7_April_2008&action=edit 7] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/8_April_2008&action=edit 8] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/9_April_2008&action=edit 9] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/10_April_2008&action=edit 10] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/11_April_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/12_April_2008&action=edit 12] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/13_April_2008&action=edit 13]
[http://2008.igem.org/EPF-Lausanne/14_April_2008 14] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/15_April_2008&action=edit 15] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/16_April_2008&action=edit 16] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/17_April_2008&action=edit 17] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/18_April_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/19_April_2008&action=edit 19] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/20_April_2008&action=edit 20]
[http://2008.igem.org/EPF-Lausanne/21_April_2008 21] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/22_April_2008&action=edit 22] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/23_April_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/24_April_2008&action=edit 24] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/25_April_2008&action=edit 25] [http://2008.igem.org/EPF-Lausanne/26_April_2008 26] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/27_April_2008&action=edit 27]
[http://2008.igem.org/EPF-Lausanne/28_April_2008 28] [http://2008.igem.org/EPF-Lausanne/29_April_2008 29] [http://2008.igem.org/EPF-Lausanne/30_April_2008 30]
May
MTWTFSS
      [http://2008.igem.org/EPF-Lausanne/1_May_2008 1] [http://2008.igem.org/EPF-Lausanne/2_May_2008 2] [http://2008.igem.org/EPF-Lausanne/3_May_2008 3] [http://2008.igem.org/EPF-Lausanne/4_May_2008 4]
[http://2008.igem.org/EPF-Lausanne/5_May_2008 5] [http://2008.igem.org/EPF-Lausanne/6_May_2008 6] [http://2008.igem.org/EPF-Lausanne/7_May_2008 7] [http://2008.igem.org/EPF-Lausanne/8_May_2008 8] [http://2008.igem.org/EPF-Lausanne/9_May_2008 9] [http://2008.igem.org/EPF-Lausanne/10_May_2008 10] [http://2008.igem.org/EPF-Lausanne/11_May_2008 11]
[http://2008.igem.org/EPF-Lausanne/12_May_2008 12] [http://2008.igem.org/EPF-Lausanne/13_May_2008 13] [http://2008.igem.org/EPF-Lausanne/14_May_2008 14] [http://2008.igem.org/EPF-Lausanne/15_May_2008 15] [http://2008.igem.org/EPF-Lausanne/16_May_2008 16] [http://2008.igem.org/EPF-Lausanne/17_May_2008 17] [http://2008.igem.org/EPF-Lausanne/18_May_2008 18]
[http://2008.igem.org/EPF-Lausanne/19_May_2008 19] [http://2008.igem.org/EPF-Lausanne/20_May_2008 20] [http://2008.igem.org/EPF-Lausanne/21_May_2008 21] [http://2008.igem.org/EPF-Lausanne/22_May_2008 22] [http://2008.igem.org/EPF-Lausanne/23_May_2008 23] [http://2008.igem.org/EPF-Lausanne/24_May_2008 24] [http://2008.igem.org/EPF-Lausanne/25_May_2008 25]
[http://2008.igem.org/EPF-Lausanne/26_May_2008 26] [http://2008.igem.org/EPF-Lausanne/27_May_2008 27] [http://2008.igem.org/EPF-Lausanne/28_May_2008 28] [http://2008.igem.org/EPF-Lausanne/29_May_2008 29] [http://2008.igem.org/EPF-Lausanne/30_May_2008 30] [http://2008.igem.org/EPF-Lausanne/31_May_2008 31]
June
MTWTFSS
            [http://2008.igem.org/EPF-Lausanne/1_June_2008 1]
[http://2008.igem.org/EPF-Lausanne/2_June_2008 2] [http://2008.igem.org/EPF-Lausanne/3_June_2008 3] [http://2008.igem.org/EPF-Lausanne/4_June_2008 4] [http://2008.igem.org/EPF-Lausanne/5_June_2008 5] [http://2008.igem.org/EPF-Lausanne/6_June_2008 6] [http://2008.igem.org/EPF-Lausanne/7_June_2008 7] [http://2008.igem.org/EPF-Lausanne/8_June_2008 8]
[http://2008.igem.org/EPF-Lausanne/9_June_2008 9] [http://2008.igem.org/EPF-Lausanne/10_June_2008 10] [http://2008.igem.org/EPF-Lausanne/11_June_2008 11] [http://2008.igem.org/EPF-Lausanne/12_June_2008 12] [http://2008.igem.org/EPF-Lausanne/13_June_2008 13] [http://2008.igem.org/EPF-Lausanne/14_June_2008 14] [http://2008.igem.org/EPF-Lausanne/15_June_2008 15]
[http://2008.igem.org/EPF-Lausanne/16_June_2008 16] [http://2008.igem.org/EPF-Lausanne/17_June_2008 17] [http://2008.igem.org/EPF-Lausanne/18_June_2008 18] [http://2008.igem.org/EPF-Lausanne/19_June_2008 19] [http://2008.igem.org/EPF-Lausanne/20_June_2008 20] [http://2008.igem.org/EPF-Lausanne/21_June_2008 21] [http://2008.igem.org/EPF-Lausanne/22_June_2008 22]
[http://2008.igem.org/EPF-Lausanne/23_June_2008 23] [http://2008.igem.org/EPF-Lausanne/24_June_2008 24] [http://2008.igem.org/EPF-Lausanne/25_June_2008 25] [http://2008.igem.org/EPF-Lausanne/26_June_2008 26] [http://2008.igem.org/EPF-Lausanne/27_June_2008 27] [http://2008.igem.org/EPF-Lausanne/28_June_2008 28] [http://2008.igem.org/EPF-Lausanne/29_June_2008 29]
[http://2008.igem.org/EPF-Lausanne/30_June_2008 30]
July
MTWTFSS
  [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
MTWTFSS
        [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
MTWTFSS
[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
MTWTFSS
    [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]
November
MTWTFSS
          [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/1_November_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/2_November_2008&action=edit 2]
[http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/3_November_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/4_November_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/5_November_2008&action=edit 5] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/6_November_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/7_November_2008&action=edit 7] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/8_November_2008&action=edit 8] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/9_November_2008&action=edit 9]
[http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/10_November_2008&action=edit 10] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/11_November_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/12_November_2008&action=edit 12] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/13_November_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/14_November_2008&action=edit 14] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/15_November_2008&action=edit 15] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/16_November_2008&action=edit 16]
[http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/17_November_2008&action=edit 17] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/18_November_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/19_November_2008&action=edit 19] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/20_November_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/21_November_2008&action=edit 21] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/22_November_2008&action=edit 22] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/23_November_2008&action=edit 23]
[http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/24_November_2008&action=edit 24] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/25_November_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/26_November_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/27_November_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/28_November_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/29_November_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=EPF-Lausanne/30_November_2008&action=edit 30]

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

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





Prepare a master mix without the library primer, and load 28L into a 96 well plate. Add 2L 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 70L 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.

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 2.5L lyse ‘n go buffer (Pierce) o optionally 2.5L 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 1L primer pair and 46.5 L PCR Mix

PCR Mix: 1L dNTP 10L 5x Buffer + MgCl2 .5L HiFi DNA polymerase 35L dH2O 46.5L 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 1L 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

1L dNTP 10L 5x Buffer + MgCl2 1L Primer Mix 0.5L template 0.5L HiFi Plus DNAp 37L dH2O 50L 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 1L 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 1L product on a 1% agarose gel • no further purification is necessary even though PCR purification is recommended, eluting in 50L of elution buffer (EB) or pH adjusted dH2O

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

Retrieved from "http://2008.igem.org/Team:EPF-Lausanne/Notebook"