Team:Nevada/Notebook

From 2010.igem.org

(Difference between revisions)

Revision as of 01:37, 11 September 2010

You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.
Team Example

Home	Team	Official Team Profile	Project	Parts Submitted to the Registry	Modeling	Notebook	Safety

Team Nevada Notebook

Week of April 11-17:

Bryson, Michael, Senny, Tyler
- Made tobacco cell (NT-1) media in Dr. Shintani's lab

Week of April 18-24:

Bryson
- EcoRI digest of pBIB
- Made Na acetate buffer

Week of April 25-May 1:

Bryson
- Ran agarose gel of EcoRI digest

Week of May 2-8:

Elaine
- Ran 0.8% agarose gel of EcoRI digest
- Made LB/KAN plates
- Spread for colonies
- Did miniprep for pBIB liquid cultures

Week of May 9-15:

Bryson
- Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.
- Did minipreps on additional pBIB liquid cultures.

Elaine
- Did XbaI and EcoRI digest of pBIB
- Ran 2 0.8% gels of each digest
- Did a miniprep and a Phenol:chloroform clean-up
- Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB

Week of May 16-22:

Bryson
- Klenow reactions of EcoRI digests
- Phenol:chloroform cleanup of pBIB prior to ligation
- Blunt-end ligation of klenowed pBIB

Randy Pares and Vidim Gladwell
- Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.

Elaine
- Made LB/KAN plates
- Made 50mg/ml stock of KAN
- Made 1X TAE buffer

Week of May 23-29:

Bryson
- Incubated 50 mL liquid culture of E. coli with pBIB (samples 3).
- Miniprepped sample 3, using modified protocol for large/low-copy plasmids.
- EcoRI digest of uncut sample 3
- Prepared 5 mM dNTP stock

Elaine
- Incubated 50 mL liquid culture of E. coli with pBIB (sample 4 & sample 5)
- Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids
- Nanodrop of DNA recovery of miniprepped sample 4 & sample 5

Randy and Vadim
- Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22
- Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene
- Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit

Week of May 30-June 5:

Bryson
- HinD3 digest of uncut sample 3
- Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI
- Pooled uncut samples 3, 4 and 5 (pBIB-pool)
- Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)
- EcoRI digest of pBIB-pool and pBIB-maxi
- Ran 0.8% agarose gel of EcoRI digests
- Klenow reactions of pBIB-pool and pBIB-maxi
- Made glycerol stocks of pBIB samples 1-5

Elaine
- EcoRI digest of uncut pBIB sample 4 and 5
- HinD3 digest of uncut sample 4 & sample 5 as a positive control
- Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful
- Ethanol precipitation of the EcoRI digests of sample 4 & sample 5
- Nanodrop resulted to a low DNA content
- Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep
- Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest
- Modified all protocols of the Binary vector

Randy and Vadim
- Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)
- Programmed thermal cycler for PCR of ccdB gene
- Ran PCR for ccdB
- Prepared LB/KAN Broth
- Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel
  - Reaction was unsuccessful

Week of June 6-12:

Bryson
- Ligation reactions for pBIB-pool and pBIB-maxi
- Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB
- Transformed Top-10 Cells with modified pBIB (designated pBIB#)
  - Obtained two colonies after overnight incubation
- Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate

Randy and Vadim
- Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI
- Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB
- Modified thermal cycler conditions for PCR of ccdB gene
- Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)
- Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene
- Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit

Week of June 13-19:

Bryson
- Prepared liquid cultures of pBIB# 1 and pBIB# 2
- Miniprepped liquid cultures of pBIB#
- EcoRI and HinDIII digests of pBIB#
- Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#
- Single-colony streaked pBIB# 2 on a fresh LB-Kan plate

Elaine
- Worked with Chris to incubate 30 liquid cell cultures
- Ran 0.8% gels of all 30 samples

Randy and Vadim
- Topocloned ccdB gene into TOPO PCR Blunt II vector
- Determined concentration of pBIB maxipreps using PicoGreen analysis
- Single-colony isolated nine colonies of TOPO-cloned ccdB gene
- Miniprepped cultures of ccdB gene in TOPO-clone
- Digested minipreps using EcoRI
- Ran 1% gel for digested minipreps
- Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9
- Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector

Week of June 20-26:

Bryson
- Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate
- Started 20 mL liquid cultures of 2-1, 2-2, and 2-3
- Miniprepped samples
- EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony
- 0.8% agarose gel of digests

Randy and Vadim
- Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit
- Nanodropped minipreps
- Digested minipreps using EcoRI
- Ran 1% gel for digested minipreps
- Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added
- Analyzed ccdB samples using Vector NTI (Invitrogen)
- Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added

Elaine
- Primer Design of RD29A

Week of June 27-July 3:

Randy and Vadim
- Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit
- Prepared thermal cycler protocol for pBIB vector
- Performed multiple PCR on pBIB vector
- Transformed ccdB into NEB cells unsuccessfully
- Made chlorophenocol resistant agar plates
- Agarose gel analyzed PCR products

Week of July 4-10:

Randy and Vadim
- Agarose gel analyzed PCR products
- Transformed ccdB into NEB, OmniMax, and DEB cells
- Made Kanamycin resistant LB plates

Elaine
- Transformed ccdB into NEB, OmniMax, and DEB cells
- Made LB/KAN plates
- Took picture of ccdB Sensitivity Experiment 1 Results
- GFP Primer Design

Week of July 11-17:

Elaine
- Made LB/Amp plates

Randy and Vadim
- TOPOcloned pBIB fragment
- Attempted to ligate ccdB into iGEM vector pSB1C3
- Single colony streaked ccdB transformation
- Made Chloramphenicol broth
- Performed PCR on pBIB cell lines
- Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI
- Miniprepped ccdB colonies
- Gel analyzed pBIB PCR product
- Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates
- Made 50x TAE buffer
- Miniprepped pSB1C3+RFP vector

Week of July 18-24:

Elaine
- PCR of GFP
- Ran agarose gel analysis of GFP PCR product

Randy and Vadim
- Amplified pBIB fragment using PCR
- Gel analyzed pBIB PCR product
- Attempted ligation of ccdB into iGEM vector pSB1C3
- Transformed ligation into NEB cells
- Selected supposed colonies with ccdB+pSB1C3

Week of July 25-31:

Elaine
- GFP Transformation
- Cell Culture of GFP
- Miniprep of GFP
- EcoR I & Pst Digest of GFP
- Ran 1.2% agarose gel analysis of the GFP digest
- PCR of GFP

Randy and Vadim
- Miniprepped ccdB+pSB1C3 colonies
- Digested minipreps with EcoRI and PstI
- Performed colony PCR on pSB1C3 colonies

Week of August 1-7:

Elaine
- Ran 1.2% Agarose Gel analysis of GFP PCR product
- Topocloned GFP PCR product
- Cell Culture of GFP Topocloned colonies
- Streaked colony plate
- Miniprep of GFP Topocloned colonies
- EcoR I digestion of GFP Topocloned
- Ligation Test: GFP original vector digested with EcoR I

Samantha and Christian
- pMA and pBIB liquid cultures and mini preps

Bryson
- Designed and ordered primers for the 35S promoter
- Made spectymycin plates
- Transformed DB3.1 cells with pK7FWG2
- Liquid cultures and minipreps to isolate pK7FWG2

Week of August 8-14:

Elaine
- Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest
- Ligation Test: Ligated EcoR I digest GFP original vector
- Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells
- Cell culture and streaked colonies of RFP in PSB1C3 vector
- Miniprepped RFP in PSB1C3 vector
- Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI
- Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors
- Ligated the RFP & GFP in either PSB1C3 & Topo Vectors
- Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates

Samantha and Christian
- Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI
- Ligation of triple digest products

Bryson
- PCR of pK7FWG2

Week of August 15-21:

Elaine
- Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates
- Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors
- Digested GFP in PSB1C3 vectors with EcoR1 & PstI
- Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI

Bryson
- 1.2% gel to confirm presence of amplicon
- Topocloning of amplicon and transformation of Topo vector into DB3.1 cells

Week of August 22-28:

Elaine
- Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)
- Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17
- Miniprepped 8 cell cultures and nanodrop
- Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)

Bryson
- Isolated and streaked 6 colonies
- Liquid cultures and minipreps of samples
- Digest of samples with EcoRI and PstI to confirm presence of insert

Week of August 29-September 4:

Elaine
- Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
- Sequencing analysis of colony #11 & #17

Bryson
- Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation
- Miniprepped cultures and eluted in 300 microliters
- 50 microliter digests done
  - 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids
  - Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction

Week of September 5-11:

Elaine
- Sequenced 4 tubes of #17 of GFP in PSB1C3
- Digested RD29A with Hind III & MfeI-HF

Bryson
- Transformation of ligation reaction
- Plated on chloramphenicol
- Selected four white colonies and single-colony streaked them
- 4 mL liquid cultures of colonies
- Miniprepped samples and eluted in 50 microliters
- 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3

Week of September 12-18:

Week of September 19-25:

Week of September 26-October 2:

Week of October 3-9:

Week of October 10-16:

Week of October 17-23:

Week of October 24-30:

@@ Line 266: / Line 266: @@
 *Samantha and Christian
 **pMA and pBIB liquid cultures and mini preps
+* ''Bryson''
+** Designed and ordered primers for the 35S promoter
+** Made spectymycin plates
+** Transformed DB3.1 cells with pK7FWG2
+** Liquid cultures and minipreps to isolate pK7FWG2
 '''Week of August 8-14:'''
@@ Line 282: / Line 288: @@
 **Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI
 **Ligation of triple digest products
+* ''Bryson''
+** PCR of pK7FWG2
 '''Week of August 15-21:'''
@@ Line 290: / Line 299: @@
 ** Digested GFP in PSB1C3 vectors with EcoR1 & PstI
 ** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI
+* ''Bryson''
+** 1.2% gel to confirm presence of amplicon
+** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells
@@ Line 298: / Line 311: @@
 ** Miniprepped 8 cell cultures and nanodrop
 ** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
+* ''Bryson''
+** Isolated and streaked 6 colonies
+** Liquid cultures and minipreps of samples
+** Digest of samples with EcoRI and PstI to confirm presence of insert
@@ Line 304: / Line 322: @@
 ** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
 ** Sequencing analysis of colony #11 & #17
+* ''Bryson''
+** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation
+** Miniprepped cultures and eluted in 300 microliters
+** 50 microliter digests done
+*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids
+*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction
 '''Week of September 5-11:'''
@@ Line 309: / Line 334: @@
 ** Sequenced 4 tubes of #17 of GFP in PSB1C3
 ** Digested RD29A with Hind III & MfeI-HF
+* ''Bryson''
+** Transformation of ligation reaction
+** Plated on chloramphenicol
+** Selected four white colonies and single-colony streaked them
+** 4 mL liquid cultures of colonies
+** Miniprepped samples and eluted in 50 microliters
+** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3
+'''Week of September 12-18:'''
+'''Week of September 19-25:'''
+'''Week of September 26-October 2:'''
+'''Week of October 3-9:'''
+'''Week of October 10-16:'''
+'''Week of October 17-23:'''
+'''Week of October 24-30:'''