Team:Cornell/Week 10

From 2011.igem.org

(Difference between revisions)
(→Wednesday, August 10)
(→Thursday, August 11)
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==Thursday, August 11==
==Thursday, August 11==
 +
Lab work done by: Charlie, Claire, Bill, Nick, and Youjin
 +
 +
:'''Objective'''
 +
::*Coat microfluidic device with steptavidin.
 +
:'''Diluting chemicals'''
 +
::*4% MPTMS in ethanol (MPTMS = 3-mercaptopropyl trimethoxysilane)
 +
:::4mL MPTMS
 +
:::96mL EtOH
 +
::*1mM GMBS in ethanol
 +
:::0.014gGMBS (280.2g/mol)(GMBS = N-g-maleimidobutyryloxy succinimide ester)
 +
:::50mL EtOH
 +
::*25ng/mL NeutrAvidin in PBS - serial dilutions
 +
:::Dilute 0.25mg in 10mL PBS = 0.025mg/mL
 +
:::Take 1mL of first dilution and bring to 10mL = 2.5ug/mL
 +
:::Take 1mL of second dilution and bring to 100mL = 25ng/mL
 +
:'''Streptavidin coating'''
 +
::*Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046
 +
::1) 45 min in 4% (by volume) MPTMS in ethanol
 +
::2) 20 min in 1mM GMBS
 +
::3) 45 min in 25ng/mL NeutrAvidin in PBS
==Friday, August 12==
==Friday, August 12==

Revision as of 19:33, 11 August 2011

Results | Protocol | Notebook | Parts Submitted

Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |


August 7th - August 13th

Sunday, August 7

Morning lab work done by: Youjin Cho and Claire Paduano

Objective
Prepare VioE in Avi-Tagged pZE12 vector backbone for VioA and VioB gene inserts
Digestion Setup
23.65μL H2O
18.6μL VioE in Avi-Tagged pZE12 vector backbone (1μg)
5μL 10x NEBuffer 2
0.5μL 100x BSA
1.25μL KpnI
1μL HindIII
50μL Total
KpnI has only 75% efficiency in Buffer 2, so added 0.25uL more KpnI to digestion mixture
Incubate in 37°C water bath for 2 hours

Afternoon lab work done by: Jim Mathew, Charlie Chung

Objective
Ligate vioA and vioB genes onto their own pZE12 vector backbone, in place of the vioE that is now digested out
Ligation Reaction Setup
  • vioA + Avi-Tagged pZE12 vector backbone
11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
3.3μL vioA gene insert (cut with KpnI & HindIII)
2.3μL H2O
2μL 10x T4 DNA ligase buffer
1μL T4 DNA ligase
20μL Total
  • vioB + Avi-Tagged pZE12 vector backbone
9μL vioB gene insert (cut with KpnI & HindIII)
8μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
2μL 10x T4 DNA ligase buffer
1μL T4 DNA ligase
20μL Total
  • Control Ligation for vioA and vioB Gene Inserts
11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
5.6μL H2O (gene insert volumes go toward water volume)
2μL 10x T4 DNA ligase buffer
1μL T4 DNA ligase
20μL Total
Incubate all ligation reaction constructs overnight in 16°C waterbath.

Evening lab work done by: Charlie Chung

Objective
Because previous DH5α colonies transformed with (vioE + Avi-Tagged backbone) are very small, in addition to one failed 5mL culture and one discarded Miniprep sample, incubated plate for four more hours.
  • Picked three new colonies. Labeled as "vioE + Avi + BB #4, 5, 6"
  • 5mL LB cultures with 5μL ampicillin incubated overnight in 37°C shaker of Room 304.

Monday, August 8

Morning lab work done by: Youjin Cho and James Mathew

Objective
  • Transform the overnight ligation of vioA/vioB + Avi-Tag + pZE12.
  • Send the Miniprepped samples (GFP, RFP, vioE) from Saturday in for sequencing.
  • Miniprep the additional vioE samples that were grown overnight.
Transformation
  • Desalted the ligation reaction samples(vioA/vioB + Avi-Tag + pZE12) on a membrane for 15 minutes.
  • After adding the samples into DH5α cell lines, electroporated them and put into shaker for 45 minutes.
  • The samples were plated on the plate with ampicilin.
Sequencing
  • Using the reverse primer dilution, the samples were sent in for sequencing.
GFP + Avi-Tag + pZE12 - 1
GFP + Avi-Tag + pZE12 - 2
GFP + Avi-Tag + pZE12 - 3
RFP + Avi-Tag + pZE12 - 1
RFP + Avi-Tag + pZE12 - 2
RFP + Avi-Tag + pZE12 - 3
vioE + Avi-Tag + pZE12 - 2
Miniprepping the vioE samples
  • The samples were Miniprepped using standard Qiagen Miniprep protocol.
vioE + Avi-Tag + pZE12 - 4
vioE + Avi-Tag + pZE12 - 5
vioE + Avi-Tag + pZE12 - 6

Evening lab work done by: Charlie Chung

  • Checked to see if colonies were ready to pick from transformed plates of (vioA + Avi-Tagged pZE12 backbone) and (vioB + Avi-Tagged pZE12 backbone).
  • vioA plate is showing early signs of a good number of colonies. Too small for picking, however.
  • vioB and Control plates look pretty empty (but just may not have grown up enough to tell).
  • Will be best if colonies were picked Tuesday (tomorrow) morning. Please find the 3 plates still in the 37°C incubator.

Tuesday, August 9

Evening lab work done by: Youjin Cho

  • Picked colonies from transformed plates of (vioA + Avi-Tagged pZE12 backbone) and (vioB + Avi-Tagged pZE12 backbone).
vioA 1,2,3
vioB 1,2,3
  • Put them into the 37°C shaker around 5:30 PM.

Wednesday, August 10

Morning lab work done by: Youjin Cho & Alyssa Henning

Objective
  • Miniprep the samples of vioA/vioB + Avi-Tag + pZE12 backbone and measure their concentrations.
  • Submit the Miniprepped samples for sequencing.
  • Since the sequencing results showed that GFP did not work, run a PCR reaction for more GFP.
Miniprepping the vioA, vioB samples
  • The samples were Miniprepped using standard Qiagen Miniprep protocol.
vioA + Avi-Tag + pZE12 - 1 = 67.6 ng/µL
vioA + Avi-Tag + pZE12 - 2 = 70.6 ng/µL
vioA + Avi-Tag + pZE12 - 3 = 71.4 ng/µL
vioB + Avi-Tag + pZE12 - 1 = 66.0 ng/µL
vioB + Avi-Tag + pZE12 - 2 = 70.6 ng/µL
vioB + Avi-Tag + pZE12 - 3 = 75.9 ng/µL
Sequencing
  • The miniprepped samples were prepared for sequencing using the diluted reverse pZE12 primer.
Order number: 10254703
1 vioA-1
2 vioA-2
3 vioA-3
4 vioB-1
5 vioB-2
6 vioB-3
PCR for GFP
  • Since the result from the sequencing showed that GFP did not work, ran a PCR reaction using the original template of GFP in pZE12.
  • Ran two reactions and labeled them 1,2 GFP igem 8/10.
1 GFP = the sample from May 5th GFP pZE12 with concentration of 50.6 ng/µL.
2 GFP = the sample without date with concentration of 20.2 ng/µL.
  • The PCR was ran with a stadard PCR setup with melting temperature of 54.2°C for 1 minute.

Thursday, August 11

Lab work done by: Charlie, Claire, Bill, Nick, and Youjin

Objective
  • Coat microfluidic device with steptavidin.
Diluting chemicals
  • 4% MPTMS in ethanol (MPTMS = 3-mercaptopropyl trimethoxysilane)
4mL MPTMS
96mL EtOH
  • 1mM GMBS in ethanol
0.014gGMBS (280.2g/mol)(GMBS = N-g-maleimidobutyryloxy succinimide ester)
50mL EtOH
  • 25ng/mL NeutrAvidin in PBS - serial dilutions
Dilute 0.25mg in 10mL PBS = 0.025mg/mL
Take 1mL of first dilution and bring to 10mL = 2.5ug/mL
Take 1mL of second dilution and bring to 100mL = 25ng/mL
Streptavidin coating
  • Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046
1) 45 min in 4% (by volume) MPTMS in ethanol
2) 20 min in 1mM GMBS
3) 45 min in 25ng/mL NeutrAvidin in PBS

Friday, August 12

Saturday, August 13

Retrieved from "http://2011.igem.org/Team:Cornell/Week_10"