Team:UoA NewZealand/WetLab
Wet Lab
Overview and Results
11/8 First Result ever: Found that the DE3 E. coli cells we were using were intrinsically resistant to chloramphenicol. Couldn’t tell using the iGEM competency test (fancy name for it?) (which added a plasmid with chloramphenicol resistance) how competent our cells were, as those with the plasmid included would have been selected against. Switched to DH5 alpha cells for further recombination.
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- Were they chemically competent?
- They were, calculations to the competency on page 12 of lab book 06/09
- Also other competent cells - 09/09
- gels/photos would be gr9
- Optional: mention the fricking RE sites in the middle of the construct?
- 16/08 (labbook) - Optional: talk about issues with ligation/understanding provided igem protocol
- Gel - 31/08
- 03/09 - 04/09 - made new chemically competent cells
- Gained PCR skills 07/09, 08/09
Cells passed competency test, but really struggled to take up our construct, switched to electroporation as recommended by our PI, and as it would remove more steps that could go wrong in chemical transformation.
23/09 Kan backbone + PETase End colonies grew on plates 1) Kan plate and competent cells - no growth 2) + 3) - PETase end + kanamycin backbone - many small colonies 4) negative control - kan plate and no cells - no growth 5) plain agar plate with cells ‘transformed’ with PETase end (to see growth of cells after transformation) - lawn of cells
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Miniprep of kan backbone + PETase end -> 27/09, gel page 20, used another miniprep protocol 29/09 - still no DNA
- Could be due to overgrowing colonies
- Unlikely, cells displayed kanamycin resistance when innoculated in higher concentration (80ng/ul vs 50ng/ul) and when grown on fresh plates
- Grew cells on someone else’s kanamycin - still grew
- Could be due to fault in construct - EcoRI site that would have cut up the PETase end
- Self-ligation of the backbone?
- Backbone insertion into the DNA?
- WHY
Decided to use fresh DH5 alpha cells and to ditch the faulty construct
03/10 Third Result: electroporation results Decided to use another backbone (pSB1C3) as gel (page 34 - 04/10) showed that PCR of kan backbone not entirely pure and was worried with the previous results of the plasmid being able to insert into the genome/being low copy number/self-ligating. Successfully transformed cells with plasmid given by one of our advisors & confirmed with miniprep page 44 - therefore transformation is not the problem
- 1 = ampicilin plate with cells transformed with peroxyredoxin plasmid- many big colonies
- 2 = kan backbone only transformants - no growth
- 3 = col backbone with PETase start -no growth
- Did some transformations with cells provided by our PI - as they were known to be electrocompetent. Also used a plasmid with a multiple cloning site we could use for our construct, pET-DUET to see whether that would take up our construct any easier.
- 4 = chloramphenicol plate with col backbone digest at 9/10 dilution (no insert) - lawn of plates - issue with antibiotic plating/spreading
- 5 = ampicilin plate + petduet + PETase - no growth
- 6 = canvas
- 7 =
3 = chlorampheicol plate with col backbone + PETase start insert -
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Forth Results: start monitoring on the digestion and ligation concentration - put on gel to see results.
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Lab Notes
Date: 25/07/16
People in lab: Jess, Grace, David, Irene, Peter Goal: Training via purifying protein for Kyle (you’re welcome) * Made filtered water for columns * Collected transformed bacteria from –80C freezer * David & Irene (afternoon) * Prepared Lysis buffer and Sec Buffer * Jess & Grace (afternoon) * Lysed cells using French press. * X * Lysate prepared for centrifugation - increase concentration - Imac column prepared. * {Didn’t run sec on AKTA}
Date: 1/8/16–3/8/16
People: Jess (? handwriting) Goal: Make chemically competent cells * Made chemically competent cells using protocol on Slack (link) * Incubated overnight * Prepared * Cacl2 (0.1M) / H20 / Glycerol 15% * CaCl2 = 0.441g * H20 = 25.5mL * Glycerol = 4.5 mL
Date: 9/08/16
Morning:
People: Jess & Grace
Goal: Ligation and Digestion {{was using old IGEM protocol - results will have failed }}
Protocol: 3A Assembly with linearised Plasmid Backbone - IGEM (link)
* 10am - Jess gone through brief
* Made up three master mixes as specified.
Afternoon: 3–4 pm
People: May & ???
Goal: Prepare Plates
* Made LB agar plates for competency testing:
* 9 x Chloramphenicol plates
* 3x 0.5 pg (picograms)
* 3x 10 pg
* 3 x 50 pg
* 20mL agar to 20uL chloramphenicol per plate (original conc?)
* 4 x kanamycin plates for transformation
* 20mL agar to 20uL kanamycin (original conc???)
* 1x agar plate w/o antibiotic (control)
Date: 11/09/16
People: Jess, David, Grace, Irene Commenced - 10am Goals: Create correct ligation products * Note: Previous protocol used for ligation incorrect, used new protocol Protocol: Protocol for 11/08 (link) * Made Enzyme Master Mix * Created 3 small bottles of Soc & autoclaved * Digested Backbone & insert (which ones????) - assuming the IGEM kan one + PETase start * Ligation carried out with digestion product, ligase buffer and t4 DNA ligase {and we all know how well that went- no ATP }
Date: 12/09/16
People: ???? (Jess?) Goals: Check transformation * Checked colonies: * Competency worked - colourless colonies {little did we know……. They were already chloramphenicol resistant} * Transformed Bacteria (PETases) - no colonies * Incubated overnight to see if would get new growth
Date: 13/09/16
People: Jess & May Goals: Check transformation - create new ligation? Jess? * Checked PETase plates * 2x100uL no colonies * 2x200uL - some colonies - yellow and white * {what does this mean someone plz} * Stored old PETase competent cells in 37C incubation overnight - check for more growth
Date: 16/08/16
People: Grace & Jess Goal: Troubleshooting™ * Ran DNA gel to see whether ligation/insertion was working * 0.9 % agarose gel with Ethididum Bromide Used * PETase insert ~ 1kbp * Plasmid ~2kbp * Gel image in book * Ran 1kb ladder * 11/8 PETase ligation (start?) * Faint band around 2kbp - plasmid only * 15/8 PETase ligation (start?) * No bands seen on gel * PETase insert (start?) * Stronger band around 1kb * Concluded that ~something~ was wrong with ligation
Date: 17/08/16
People: David * Nanodrop spec on plasmid backbone (which one - IGEM kan?) * 32.05ng ul–1 concentration
Date: 29/08/16
People: David? * Followed 19th August protocol for Plasmid construction. Have completed digestion and ligation steps * Made 500mL Agar * Plates to be spread are stored in second drawer at lab bench * Problem : Accidentally had 6 minutes of incubation at 37C after heat kill cycle. This happened during digestion. * 0.8ul of T4 DNA ligase buffer added + 3.2 ul of water. (1:5 dilution) * ATP ? 1/10 concentration - was ACTUALLY ? 1/11 concentration * ATP stock at 10mM needed at 0.1 mM
Date: 30/08/16
People: David, Irene, Jess
May & Peter joined in later
* Carried out PETase (Start & End) transformations. Also conducted competency test.
* Used protocol Protocol for 30/08)
* Alterations;
* Original iGEM competency plasmid concentrations used as well as serial dilution, plasmids at concentrations of (ng/uL):
* 50, 20, 10, 5, 09, 5x10^–2, 5x10^–3, 5x10^–4, 5x10^–5
* Cells incubated for 2 hours rather than 30 minutes prior to plating
* Shaken at 180rpm
* Plated out transformed cells (and competency)
Date: 31/08/16
People: David, Thai & Irene (10am) * Plates collected from incubator * All competency plates have high growth * DE3 strain already has Col resistance * No growth on PETase plates * Ran gel with plasmid constructs * Gel on page 10 of lab book * (no notes on gel, will summarise here) * Appeared to be no ligation products for Start, ligation for End showed faint band at ~100 kbp
Date: 01/09/16
People: Irene & David Protocol: 01/09 protocol * Made up stock of LB & glycerol * Plated DH5αα alpha cells on kanamycin and chloramphenicol to check if resistance already present. Plain agar control. * Prepped for making electrocompetent cells: seeded culture of DH5αα alpha overnight.
Date: 02/09/16
People: Thai & Jess Protocol : 02/09 protocol The day things fucked up :’< * Didn’t make up chemically competent cells - ran out of time everything had to be chucked out
Date: 03/09/16 - 04/09/16
People: Maggie * Made chemically competent cells * Used protocol from Shaun’s Lab guide (found in lab folder)
Date: 05/09/16
People: Irene, David & May Protocol: 05/09 protocol * DH5αα alpha cells from Maggie were transformed with plasmids for PETase from previous week’s ligation. * Competency test also carried out * Incubated cells
Date: 06/09/16
People: Irene & then May * Made up LB and glycerol * Results of Competency and Transformation * No growth of PETase start or end (on antibiotic?) * Plain agar plates with PETase cells didn’t grow either * Competency test successful * Lots of calculations to calculate competency of maggie’s cells (page 12)
Date: 07/09/16
People: David & Jess * Ran PCR of PETase start, end and IGEM kanamycin backbone * Original concentrations of PETase start and end (nanodrop) * PETase start - 19.96 pg/uL (average) * PETase end - 31.73 pg/uL (average)
Date: 08/09/16
People: Irene * Ran gel of PCR products (page 14) * Ladder leaked into neighbouring well * Showed there was PCR product of the inserts - backbone not as successful * Contamination of buffer - faint smear at longer bp lengths * Nanodroped concentrations - unsuccessful * (blanked with control - would have DNA primers and dNTPs so wouldn’t be the best blank)
Date: 09/09/16
People: David * DH5ααalpha competency - chemically competent cells? * Measured OD600 of 0.3 - returned to incubation room to bring to OD600 of 1 * PCR products: * Made new buffer to blank nanodrop with (what was it)?? * Machine gave warning that absorbance too high in blank * Measured concentrations in book - page 15 * Prepared to run gel - 8 lanes but ran out of time
Date: 20/09/16
People: Irene * Nanodropped PCR products - confirm DNA concentration * PETase start: 19.8ng/uL * PETase end: 18.3 ng/uL * PETase end: 18.450 ng/uL * Ran gel of PCR products - page 17 * Shows start and end have been PCR’d successfully (yay) * PCR protocol in drawer, changes: * PCR was made to volume of 25uL - with 1uL of construct and 1.5uL of each primer * Extension time = 70 seconds * Annealing temp = 65C * Transformed cells with PETase start and End * David’s cells and Maggie’s cells
21/09/16
People: David, Irene & Thai ???/ PETase - no growth on Kan plates, put back in incubator in case any transformed cells were slow growers. Kan backbone transformation had lawn of growth.?
Date: 22/09/16
People: Jess + Grace (morning) * Made up stocks of LB agar * One colony from previous transformation potentially successful * Was red - contaminant from competency test? * Plated out colony onto plates with and without kanamycin * Afternoon? -> Undertook digestion and ligation of PETase start + end
Date: 23/09/16
People: Irene, Grace, Thai & Maggie Goal: Electrotransformation funtimes Protocol: Electrotransformation as put on slack * Tried transforming PETase start and end ligation products into our electrocompetent cells * Used 2uL of ligation product - PETase start arced, but PETase end didn’t * Incubated transformed PETase end bacteria in tube at 37C for 1 hour * Made up plates for plating (extra were made, those used are below) * 20mL of agar to 20uL of kanamycin (antibiotic plate) * At end of incubation, centrifuged transformed bacteria to resuspend in agar * Very small pellet observed -few bacteria * Plated as follows * 1 plate without kan and without transformants - negative control * 1 plate without kan + plated with PETase end * 1 plate with kan + plated with PETase end * 1 plate without kan + plated with DH5ααalpha (?) * 1 plate with kan + plated with DH5αα alpha (?)
Date: 24/09/16
People: Maggie: * Checked transformed plates * No growth on negative controls * Small colonies on kan + PETase end plate - incubated a little longer to try and grow colonies a little longer
26/09/16
People: Maggie, Jess, David + Grace * PETase end grew (yay) * Maggie and Jess started miniprep following kit instructions * Miniprep yielded no results - appears to be no Plasmid present in PETase end cells
28/09/16
People: Jess, Grace + Irene - morning * Streaked out 4 colonies from PETase end transformation onto kan plates * Afternoon: Maggie + Jess * Made liquid broth cultures of 4 colonies for miniprep * Previous miniprep DNA too low to show up on gel * Gel on page 20
Date: 29/09
People: Maggie + Grace (morning) Protocol: Maggie’s miniprep * Tried different miniprep protocol of PETase end colonies grown previous day * Left the streaked colonies from previous day in incubation room to grow more. * Carried out miniprep * DNA concentration of plasmid via nanodrop non-existent * (~–7.5 ng/uL to –0.5 ng/uL) * Decided to up concentration of antibiotic to put more selective pressure on bacteria with plasmid * Made up 6 liquid cultures with 80ug/mL kanamycin (up from previous plates/liquid cultures - 50ug/mL) * Left in incubation room overnight for miniprep next day
Date: 30/09/16
People: David (???) * Miniprepped the PETase end liquid cultures that were grown with higher kanamycin concentration * Again, no results, no plasmid DNA * The start of Troubleshooting™ v 2.0 * Want to transform with: * Empty IGEM plasmid * New plasmid + Construct * Transformation with Kyle’s plasmid - peroxiredoxin * Plate transformed cells onto plates with Kyle’s kanamycin * Plated up cells onto Kyle’s kanamycin plates: * 4 PETase end on kyle’s kan plates * 1 DH5αα alpha (no transformation) plated on kyle’s kan * 1 control of plain agar, spread with DH5αα alpha (no transformation)
Date: 1/10/16
People: May, David & Grace * Created new digestion mixtures * Realised all PETase constructs except start have second EcoRI site (and Not 1 sites) * Only transform PETase start with EcoRI - will have to use XbaI for other constructs * Decided to do transformations with kanamycin backbone and chloramphenicol backbone (pSB1C3) (kan was running low on stock and why tf not at this point in time lmao) * pSB1c3 - has chloramphenicol resistance, same RE sites, and pUC19 derived ori * Digest: * Mastermix - may have extra BSA * Excess NEB in tube after adding to master mix - not sure why * Created three digests: * 1 - 6uL water / 2 uL kan backbone / 4 uL MM * 2 - 6uL PETase start (PCR) / 2uL chlor backbone / 4 uL MM * B - 4 uL MM / 4 uL water * Ligation: * Created 3 ligation products: - note, forgot to add ATP again like idiots :P * 1 - 4uL digest 1 / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 4.5 uL water * 2 - 4uL digest 2 / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 4.5 uL water * B - 4 uL digest B / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 6.5 uL water * Incubated - 16C/30 mins + 80C/20 mins * Prepped plates for spreading on transformants * 3 x kanamycin plates * Kanamycin backbone only transformants (1) * 3 x chloramphenicol plates * Chloramphenicol backbone + PETase start transformants (2) * 3 x ampicillin plates * Peroxiredoxin plasmid transformation (kyle’s) * Agar had started to set when we were plating - ampicillin will be at a slightly lower concentration * 4 x plain agar plates * 1 x each for each transformant + 1 for control * Electrotransformation * (Grace had to leave, David remained behind after brief walk through transformation protocol) * Used Shaun’s electrotransformation protocol * Transformations: * Kanamycin backbone only: * 40uL our cells + 1uL ligation 1 * kV = 1.82 / T = 6.10 ms * Peroxiredoxin: * 40uL our cells + 0.5uL plasmid (to conserve) * kV = 1.80 / t = 6.10 ms * PETase (diluted- how dilute?) ( to try and prevent arcing): * 40uL our cells + 1uL ligation product 2 * kV + t not taken * Incubated for 1 hour in 37C * Plated for overnight culture: * Ampicillin agar x 3: * 2 x original concentration of peroxiredoxin plasmids * 1 x spun and resuspended cells * Expect: colonies, if not = cells/transformation not viable * Kanamycin agar x 3: * 2 x original concentration of kanamycin backbone transformants * 1 x spun and resuspended cells * Expect: no colonies, if not = backbone can be expressed without an insert * Plain Agar + Kan backbone: * 2 x original concentration of kanamycin backbone transformants * 1 x spun and resuspended cells * Expect: colonies, if no colonies are present, then the cells/transformation is not viable * Col Agar + PETase Start x3 : Col Agar + Diluted PETase Start * If colonies are present : Successful transformation of Col resistance at least. Complete mini-prep to extract the plasmid. Colonies should be white. * If no colonies are present : Cells, transformation or ligation are erroneous * Any red colonies have the Col backbone but no insert. Any white colonies have an insert disrupting the RFP. * Plain Agar + PETase Start + Dilution x1 * Expect: colonies, if no colonies are present, then recheck everything!
Date : 2/10/2016
People : David and Grace
To do today : * Gel * Nanodrop * PCR
Gel set with EtBr * 3g Agarose * 30mL Buffer * 2uL EtBr
Nanodrop
| Result | |
| Blank with Ligation Buffer PETase Ligation Product | 195.05 (Weird! Plasmid concentration 25 uL) 194.70 |
| Blanked with DiH2O Primers: Anti | 89.15 ng/uL 88.80 ng/uL |
| Primers : Pre | 91.3 ng/uL 90,95 ng/uL |
Gel to Run
| 2 Start | ||
| Start | ||
| End | ||
| B | ||
| Buffer (LB) | ||
| Skipped | ||
| L2 PETase Ligation product | ||
| Went into 2 wells ^ | ||
| PETase Insert | ||
| PETase Digestion product | 5uL | |
| Col Backbone | 2.9 uL backbone, 1uL dye | |
| Skipped Lane | ||
| Ladder | 6uL |
WE! FORGOT! TO! ADD! ATP! (to ligation)
Gel shows that ligation didn’t work (no ATP lmao)
Date: 3/10/2016
People : ??
To run Experiment: Into cells of known competency (given by Shaun)
- Into our cells
- PETase start
- Col Backbone + Insert
- PET duet + Insert
- In Shaun’s cells
- Digested Col backbone
- Peroxiredoxin plasmid
- In both cells
- Peroxiredoxin plasmid
Bluescript could not be found, so PET duet was used instead, given by Gk.
PET duet requires EcoRI / XBaI and PSTI
PCR of PETase Start + PETase End
- PCR Mix
- KAPA Hot Start Ready Mix 25 uL
- Primer (Forward) 1.5 uL
- Reverse 1.5 uL
- Template DUT (?) 0.25 uL (6.5 ng of template)
- Water 21.75 uL
- Total 50 uL
x3 for each of PETase Start and PETase End - Tubes are labelled PCR E and PCR S
- Initial Denaturation 95 C, 3 minutes
- Denaturation 98 C, 20 seconds }
- Annealing 65 C, 15 seconds } 25 cycles
- Extension 72 C, 20 seconds }
- Final Extension 72 C, 1 minute
Nanodrop of PCR Products
NB / Peroxy Plates : restreak and see if they grow
—— table image here —–
* Going to store in iGEM fridge as is (10x concentration)
* Labelled PCRS 1-3 = Start PETase
* PCRE 1-3 = End PETase
* Approx 48 uL left in tubes (before transformation)
* Run gel of PCR products
1 uL PCR product 1 uL Loading Dye 4 uL of MQ Water
PCR PETase Start seems to have 3 bands 1 @ 1Kbp 1 @ 700 bp 1 @ 500 bp
Digestion
NEB Buffer diluted x 10
- Master Mix :
- 200 uL NEB
- 2 uL BSA (possibly ola?)
- 2 uL ECO RI
- 2 uL PstI
- 74 uL Water
- Digest:
- Col backbone + PETase Start (d KP)
- pET Duet + PETase Start (d CP)
- Col backbone (d C)
- Just MM + Water ( d MM)
- (Out of Kan backbone)
d CP : 4 uL MM, 6 uL PETase Start, 2 uL Col backbone
d PP : 4 uL MM, 6 uL PETase Start, 2 uL Pet Duet
d C: 4 uL MM, 6 uL DI H2O, 2 uL Col backbone
d MM : 4 uL MM, 8 uL DI H2O | 12 uL
- Digestion was Run :
- 37 C / 30 minutes digest
- 80 C / 20 minutes heat kill
- Sat @ 15 C for 10 minutes or so
Yesterday’s Peroxidoxin cells have been streaked onto a new plate spread with Amp.
Date : 04/10/2016 People : Jess and Grace
- Going to ligate Digest products of yesterday
- Diluted T2 ligation buffer stock
- 3 uL 10x conc. T4 ligation buffer
30 uL 1 x concentration T4 ligation buffer - must add separately
LCP
4 uL dCP 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ WaterLPP 4 uL dPP 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water
LC 4 uL dC 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water
LM 4 uL dMM 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water
BBPCR - For backbone PCR (Kanamycin backbone) 25 uL Hotstart KAPA ReadyMix 10 mM 1.5 uL Forward Primer } Stocks are 10x concentration 10 mM 1.5 uL Backward Primer }Kan Backbone —> MQ Water 21.5 uL
Ligation Product Conc.
LCP 2564.5 ng/uL LPP 1377.60 ng/uL LC 971.55 ng/uL LM 1174.3 ng/uL
~ 11:30 a.m., ran PCR on Kan backbone
Electroporation Irene, David & May came in
Gel of Ligation Products : —– table image here —–
Prepared antibiotic dilutions for transformation plates
Antibiotic stock solutions 1000x concentration (50 mg/mL) Working Concentration (50 ug/mL) Dilution -> 1uL antibiotic stock 1 mL MQ water / ethanol
- Kanamycin
- Chloramphenicol
- Ampicillin
Washing Electroporation Cuvettes -> Water, ethanol, hydrogen peroxide
Gel of Digested Products
—– table image here —–
{DNA Gel picture)
Put back in to keep running (2x DNA Gel Pictures)
Transformation: 1. Col backbone + PETase (ECP) 2. PET DUET + PETase (EPP) 3. Col backbone (EC) 4. Peroxidoxin (Prx) (in tube of conc. 1ng/uL labelled PRX)
Transformed cells :
Shaun’s DH5α, Stats unknown, Incubation began at 5:45 CP PP C Prx
iGEM DH5ααα
CP (2.49 kV, 6.10 ms) PP (2.49 V, 5.80 ms) C (2.49 V, 6 ms) Prx (Unknown)
Have all been plated up: Shaun’s on 1/10 & 9/10 plates, our iGEM cells just 100% on a plate. Restreak of peroxyredoxin cells (Kyle)
Date: 05/10/2016 People : Grace (morning)
Checked transformants Small colonies on peroxyredoxin plates (yay) Lawn of cells on chloramphenicol backbone only plates (chloramphenicol was diluted with water, this may have affected its growth) Other plates = no colonies Use Shaun’s cells from now on, our DH5ααα are probably too dilute to use
Results : Col backbone (negative control) = lawns of cells Col backbone + PETase = N/A PETDUET + PETase = N/A Peroxiredoxin (positive control) = ~100 colonies
Col has been plated on agar with water to dilute instead of ethanol, BUT no biggies .. cuz Col backbone + PETase has nothing growing on it….
Need to miniprep peroxidoxin to figure out whether the steps after ligation have been right
Why are stuffs growing on negative control? Hell knows Shaun can’t figure it out either
Improvement for next digestion : Separate backbone and insert in different tubes for digestion
New protocol : Day plans not used anymore but updates Standard protocol folder set up
To do :
Run minipreps of colonies on plates - Peroxiredoxin cells - Col backbone LB inoculated 4x Col, 2x Prx Do digest of stocks Col backbone Col + Insert Kan Kan + Insert pET DUET pET DUET + Insert Buffer
Master Mix has been made
Date : 06/10/2016 People : Jess, Grace, Irene
Following Digestion / Ligation protocol 06/10 Digesting insert + backbones separately 3x PETase Start - “PET” 1 x each backbone K = Kanamycin C = chloramphenicol D = pET Duet
Diluted PETase start PCT tube 1 as for digestion concentration needs to be ~20 ng/uL Original = 290.42 ng/uL 1/10 dilution = 29.04 ng/uL 8mL left
Kan backbone (iGEM) PCR Nanodrop
332.95 ng/uL
333.15 ng/uL
332.70 ng/uL
Diluted 1/10 to give ~ 33.2 ng /uL → 16 uL left
Miniprep liquid cultures haven’t grown - left in incubation room. Make up 3x new preparations of our peroxi transformants with 1% amp in LB Make up 3x Kyle’s peroxi transformants with 1% amp in LB 15 uL amp -> 15mL LB Kyle’s amp is already diluted - don’t use our diluted stock. Started incubation at 11:00 a.m
Creating 6 Ligation Products
LPD 1 uL of digest pET DUET 3 uL of PETase Start digest I uL ligation buffer, 0.5 uL ligase 3.5 uL of MQ Water , 1uL ATP
Assume buffer, ligase, ATP and MQ is same for next ligations if not changed
LPC 1 uL digest chloramphenicol backbone 3uL of PETase Start digest
LPK 1 uL digest kanamycin backbone 3uL of PETase insert
LD 4 uL pET DUET
LC 4 uL chloramphenicol backbone
LK 4 uL kanamycin backbone - negative control
Making up big mix of ligation mix 24.5 uL MQ Water 7 uL ligation buffer 3.5 uL T4 DNA ligase 7 uL ATP 42 uL total - 6uL into each tube
Washed electroporation cuvettes 100 % ethanol x2 washes Make sure they are dry for transformation
David made 1L of glycerol in prep. For making electrocompetent cells tomorrow
7 Transformations PK (Kan backbone + PETase) DP (Duet + PETase) CP (Chlor. Backbone + PETase) K (Kan backbone) → Neg. Control Prx (Peroxiredoxin Plasmid) → Pos. Control D (Duet backbone) → Neg. Control C (Chlor backbone) → Neg. Control Pn (pET DUET - not digested → Pos. Control
Each of the 8 transformation tubes have been filled with: 40 uL Shaun’s DH5αα cells (except Pn which had a little less) 2 uL of DNA product (except for Prx, which had 1 uL)
Transformation: —– table image here —– Began incubation at 37C @ 300rpm at 5:02 p.m.
Gel of Digest & Ligation Products
—– table image here —–
Meanwhile: 2x 10mL LB bottles inoculated with DH5α 1 plate streaked with DH5α (LB Agar)
Gel removed at ~ 8:15 & imaged (Picture)
Date: 07/10/2016 People: Jess, Grace & David
Results from yesterday’s transformations: Growth on : pET DUET without ligation Peroxiredoxin transformants (small)
Growth on outer edges of some plates Antibiotic spreading errors Kan backbone (both) Kan backbone + insert (both) Col bckbone + insert (1/10th) pET Duet without ligation (9/10th)
Potential growth on : PET DUET with PETase insert - 9/10th Faint lawn of cells Going to restreak out for cells forming faint lawn on PET DUET on plates with infused ampicillin 1x w/ amp no plating 2 x w/ amp + streaked colonies 1 x w/o amp + no plating 1 x w/o amp + streaked colonies 15 mL agar to plate → 15 uL amp (not dilute) Incubated in 37C incubator in our lab!!!!
Grace’s miniprep liquid cultures of KYle’s and our peroxiredoxin transformants has grown - ready for miniprep
Miniprep time - Grace & David Started miniprep with liquid stock of Kyle’s successful peroxiredoxin + our transformed peroxiredoxin (Shaun’s cells) Mainly to check MAggie’s protocol works + to see that we can miniprep Miniprep of iGEM 1 + 2 are mixed. Should be fine, all from same colony. Miniprep protocol - no visible DNA pellet - continued on anyway
Nanodrop Results
—– table image here —–
David restarted miniprep with “super easy plasmid extraction” (?) Visible pellets formed - looks positive
Nanodrop results (blanked with TE)
—– table image here —–
Date: 09/10/2016
People: David, Grace, Irene & Jess Transformation & Digestion: Transformed cells with: Kan backbone Kan + PETase start Col backbone Col + PETase start pET DUET pET DUET + PETase start Peroxiredoxin Digest buffer Ligation buffer Digested - seperately with 2 enzymes each to improve efficency Kan backbone Col backbone petDUET PETase Innoculated 6x seperate pET DUET PETase insert (from 7/10) colonies for miniprep
Date: 10/10
People: Irene & Grace (morning) 1 miniprep innoculum grew Digest pET DUET with single RE - see efficency ECORI, PSTI & XBAI
