Team:Nevada/Notebook

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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

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 PSBIC3 vector
- Miniprepped RFP in PSBIC3 vector

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

Week of August 15-21:

Week of August 22-28:

@@ Line 264: / Line 264: @@
 ** Ligation Test: GFP original vector digested with EcoR I
-*"Samantha and Christian"
+*Samantha and Christian
 **pMA and pBIB liquid cultures and mini preps
@@ Line 275: / Line 275: @@
 ** Miniprepped RFP in PSBIC3 vector
-*"Samantha and Christian"
+*Samantha and Christian
 **Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI
 **Ligation of triple digest products