Team:Cornell/Week 11

From 2011.igem.org

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<font size="5"> <i> August 14th - August 20th </i></font>
<font size="5"> <i> August 14th - August 20th </i></font>
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-
==Sunday==
+
==Sunday, August 14==
 +
Afternoon lab work done by: Charlie Chung
 +
:*Picked three colonies from the two (GFP + AviTagged pZE12 backbone) plates
 +
::- GFP + AviTag + BB (1) #1, 2, 3
 +
::- GFP + AviTag + BB (2) #1, 2, 3
 +
::*Incubating overnight in 37°C shaker of Room 304
 +
:*Re-picked three colonies from the (RFP + AviTagged pZE12 backbone) plate in order to retry subculturing, induction, and cell lysis
 +
::- RFP + AviTag + BB #7, 8, 9
 +
::*Incubating overnight in 37°C shaker of Room 304
-
==Monday==
+
==Monday, August 15==
 +
Afternoon lab work done by: Youjin Cho, Maneesh Gupta
 +
:'''Objective'''
 +
::*Miniprep GFP samples that were cultured overnight and send them off for sequencing
 +
::*Set up the PCR for VioB using the Vio operon
 +
:'''Miniprep & Sequencing'''
 +
::*Miniprepped the GFP samples using standard Qiagen Miniprep protocol
 +
:::GFP + AviTag + pZE12 backbone (1)- 1,2,3
 +
:::GFP + AviTag + pZE12 backbone (2)- 1,2,3
 +
::*Set up the samples for sequencing using the reverse primer
 +
:::<u>Order Number</u>: 10254977
 +
:'''PCR Reaction'''
 +
::*Set up the VioB PCR using the Vio operon (= 20.3ng/μL).
 +
::*As Didi suggested, used the melting temperature of 53°C and annealing time for 3:30 minutes.
 +
----
 +
Evening lab work done by: Maneesh Gupta, Charlie Chung
 +
:'''Preparing a Subculture'''
 +
::* Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Sunday evening
 +
:::- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture
 +
:::- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer
 +
::::* Control: 1000µL LB
 +
::::* RFP Sample: 900µL LB + 100µL (RFP + AviTagged pZE12) Colony #9
 +
::* RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Sunday's 3mL culture tube (after undoing the 1:10 dilution)
 +
::* Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture
 +
::::(3.29)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)
 +
::::? = 380µL Sunday's RFP bacteria culture to 25mL LB + 25µL ampicillin
 +
::* Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes (6:30pm start)
 +
::* At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
 +
::::OD was 0.49 at 9pm. Let culture grow until 11pm for induction (IPTG addition).
 +
::* Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at 11pm)
 +
::* Incubate induced 25mL culture flask on room temperature shaker
-
==Tuesday==  
+
==Tuesday, August 16==
 +
Afternoon lab work done by: Maneesh Gupta, Charlie Chung
 +
:*Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday.
 +
::- However, after centrifuging, the pellet was not red
 +
:'''Troubleshooting Possibilities'''
 +
::*RFP being expressed in such little quantity that it cannot be confirmed by the naked eye
 +
:::- Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for biotin
 +
::*Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long
 +
:::- Redesign primers for the sake of cloning and expressing RFP
 +
:'''What We Did'''
 +
::- Picked a culture sample from the pellet formed after the centrifuge step
 +
::- Incubated in the 37°C shaker of Room 304 for Miniprep and sequencing tomorrow
 +
:::'''p.s. -- Submit Didi's sequencing samples along with RFP sample!'''
-
==Wednesday==
+
==Wednesday, August 17==
 +
Morning lab work done by: Youjin Cho, Charlie Chung
 +
:'''Miniprep DNA Plasmid Purification'''
 +
:*Miniprepped culture from bacterial cell pellet of (RFP + AviTagged pZE12)
 +
::- Followed standard Qiagen Miniprep protocol
 +
::- NanoDrop spectrophotometry: 52.7ng/μL
 +
:'''Preparation for DNA Sequencing Submission'''
 +
::- 1μL reverse primer for pZE12
 +
::- 17μL Miniprep-purified (RFP + AviTagged pZE12)
 +
::*<u>Order Number</u>: '''10255141'''
-
==Thursday==
+
==Thursday, August 18==
-
==Friday==
+
==Friday, August 19==
 +
Lab work done by: Claire Paduano, Youjin Cho
-
==Saturday==
+
:'''Objective'''
 +
::*PCR clean up VioB insert
 +
::*Digest VioB insert and VioE in AviTagged pZE12 vector backbone and gel purification
 +
::*GFP sequencing came back: unsuccessful. PCR off GFP overnight to try ligation again
 +
:'''Digestion Setups'''
 +
::*VioE in AviTagged pZE12 vector backbone
 +
:::19.05μL H2O
 +
:::23.2μL VioE in AviTagged pZE12 vector backbone (2μg)
 +
:::5μL 10x NEBuffer 2
 +
:::0.5μL 100x BSA
 +
:::1.25μL KpnI
 +
:::<u>1μL HindIII</u>
 +
:::50μL Total
 +
::*VioB insert
 +
:::38.25μL H2O
 +
:::4μL VioB(1μg)
 +
:::5μL 10x NEBuffer 2
 +
:::0.5μL 100x BSA
 +
:::1.25μL KpnI
 +
:::<u>1μL HindIII</u>
 +
:::50μL Total
-
__NOTOC__
+
::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
 +
 
 +
:'''Gel Purification of VioB Insert and AviTagged pZE12 Vector Backbone'''
 +
::*VioB: 5.4ng/uL
 +
::*AviTagged backbone: 11.4ng/uL
 +
 
 +
==Saturday, August 20==
 +
Morning lab work done by: Claire Paduano
 +
:'''Objective'''
 +
::*Set up ligation of VioB in AviTagged backbone
 +
:'''Ligation'''
 +
::*''VioB + AviTagged Backbone''
 +
::::2.3μL AviTagged pZE12 vector backbone
 +
::::14.7μL VioB
 +
::::0.0μL H2O
 +
::::2.0μL T4 DNA ligase buffer
 +
::::<u>1.0μL T4 DNA ligase</u>
 +
::::20μL Total
 +
::In all above reactions, volume of plasmid vector added corresponds with 100ng backbone
 +
::Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs
 +
:::*Same volume of vector backbone, buffer, ligase
 +
:::*Volumes of all inserts (i.e. gene) go toward volume of H2O
 +
::After ligation setup, incubate at room temperature for 4 hours
 +
 
 +
Afternoon lab work done by: Youjin Cho, Charlie Chung
 +
:'''VioB Transformation'''
 +
::*Desalted the (VioB + AviTag + BB) ligation reaction product and the AviTagged backbone control
 +
::*Transformed (VioB + AviTag + BB) and the (AviTagged BB) control into DH5α bacteria via electroporation
 +
::*Plated onto (LB + ampicillin) dishes and incubated overnight at 37°C
 +
:'''Alternative Method for RFP Expression'''
 +
::*Sequencing results from Wednesday's (August 17) sample show that the RFP gene was present in the AviTagged pZE12 vector backbone
 +
::*Transformed 1.5μL of (RFP + Avi-Tag + BB) Miniprep-purified plasmid into DH5α bacteria with the IPTG gene via electroporation
 +
:::- Ideal Results: Colonies that have grown tomorrow will be red because RFP-production is constitutively on
 +
::*Plated onto a (LB + ampicillin) dish and incubated overnight at 37°C

Latest revision as of 17:23, 18 September 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 14th - August 20th

Sunday, August 14

Afternoon lab work done by: Charlie Chung

  • Picked three colonies from the two (GFP + AviTagged pZE12 backbone) plates
- GFP + AviTag + BB (1) #1, 2, 3
- GFP + AviTag + BB (2) #1, 2, 3
  • Incubating overnight in 37°C shaker of Room 304
  • Re-picked three colonies from the (RFP + AviTagged pZE12 backbone) plate in order to retry subculturing, induction, and cell lysis
- RFP + AviTag + BB #7, 8, 9
  • Incubating overnight in 37°C shaker of Room 304

Monday, August 15

Afternoon lab work done by: Youjin Cho, Maneesh Gupta

Objective
  • Miniprep GFP samples that were cultured overnight and send them off for sequencing
  • Set up the PCR for VioB using the Vio operon
Miniprep & Sequencing
  • Miniprepped the GFP samples using standard Qiagen Miniprep protocol
GFP + AviTag + pZE12 backbone (1)- 1,2,3
GFP + AviTag + pZE12 backbone (2)- 1,2,3
  • Set up the samples for sequencing using the reverse primer
Order Number: 10254977
PCR Reaction
  • Set up the VioB PCR using the Vio operon (= 20.3ng/μL).
  • As Didi suggested, used the melting temperature of 53°C and annealing time for 3:30 minutes.

Evening lab work done by: Maneesh Gupta, Charlie Chung

Preparing a Subculture
  • Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Sunday evening
- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture
- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer
  • Control: 1000µL LB
  • RFP Sample: 900µL LB + 100µL (RFP + AviTagged pZE12) Colony #9
  • RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Sunday's 3mL culture tube (after undoing the 1:10 dilution)
  • Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture
(3.29)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)
? = 380µL Sunday's RFP bacteria culture to 25mL LB + 25µL ampicillin
  • Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes (6:30pm start)
  • At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
OD was 0.49 at 9pm. Let culture grow until 11pm for induction (IPTG addition).
  • Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at 11pm)
  • Incubate induced 25mL culture flask on room temperature shaker

Tuesday, August 16

Afternoon lab work done by: Maneesh Gupta, Charlie Chung

  • Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday.
- However, after centrifuging, the pellet was not red
Troubleshooting Possibilities
  • RFP being expressed in such little quantity that it cannot be confirmed by the naked eye
- Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for biotin
  • Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long
- Redesign primers for the sake of cloning and expressing RFP
What We Did
- Picked a culture sample from the pellet formed after the centrifuge step
- Incubated in the 37°C shaker of Room 304 for Miniprep and sequencing tomorrow
p.s. -- Submit Didi's sequencing samples along with RFP sample!

Wednesday, August 17

Morning lab work done by: Youjin Cho, Charlie Chung

Miniprep DNA Plasmid Purification
  • Miniprepped culture from bacterial cell pellet of (RFP + AviTagged pZE12)
- Followed standard Qiagen Miniprep protocol
- NanoDrop spectrophotometry: 52.7ng/μL
Preparation for DNA Sequencing Submission
- 1μL reverse primer for pZE12
- 17μL Miniprep-purified (RFP + AviTagged pZE12)
  • Order Number: 10255141

Thursday, August 18

Friday, August 19

Lab work done by: Claire Paduano, Youjin Cho

Objective
  • PCR clean up VioB insert
  • Digest VioB insert and VioE in AviTagged pZE12 vector backbone and gel purification
  • GFP sequencing came back: unsuccessful. PCR off GFP overnight to try ligation again
Digestion Setups
  • VioE in AviTagged pZE12 vector backbone
19.05μL H2O
23.2μL VioE in AviTagged pZE12 vector backbone (2μg)
5μL 10x NEBuffer 2
0.5μL 100x BSA
1.25μL KpnI
1μL HindIII
50μL Total
  • VioB insert
38.25μL H2O
4μL VioB(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
Gel Purification of VioB Insert and AviTagged pZE12 Vector Backbone
  • VioB: 5.4ng/uL
  • AviTagged backbone: 11.4ng/uL

Saturday, August 20

Morning lab work done by: Claire Paduano

Objective
  • Set up ligation of VioB in AviTagged backbone
Ligation
  • VioB + AviTagged Backbone
2.3μL AviTagged pZE12 vector backbone
14.7μL VioB
0.0μL H2O
2.0μL T4 DNA ligase buffer
1.0μL T4 DNA ligase
20μL Total
In all above reactions, volume of plasmid vector added corresponds with 100ng backbone
Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs
  • Same volume of vector backbone, buffer, ligase
  • Volumes of all inserts (i.e. gene) go toward volume of H2O
After ligation setup, incubate at room temperature for 4 hours

Afternoon lab work done by: Youjin Cho, Charlie Chung

VioB Transformation
  • Desalted the (VioB + AviTag + BB) ligation reaction product and the AviTagged backbone control
  • Transformed (VioB + AviTag + BB) and the (AviTagged BB) control into DH5α bacteria via electroporation
  • Plated onto (LB + ampicillin) dishes and incubated overnight at 37°C
Alternative Method for RFP Expression
  • Sequencing results from Wednesday's (August 17) sample show that the RFP gene was present in the AviTagged pZE12 vector backbone
  • Transformed 1.5μL of (RFP + Avi-Tag + BB) Miniprep-purified plasmid into DH5α bacteria with the IPTG gene via electroporation
- Ideal Results: Colonies that have grown tomorrow will be red because RFP-production is constitutively on
  • Plated onto a (LB + ampicillin) dish and incubated overnight at 37°C
Retrieved from "http://2011.igem.org/Team:Cornell/Week_11"