Team:Goettingen/NoteBook w2

Observation of Back-up plates

• Part 1, C1 and C2 probably very light pink
• Part 2, C2 and C3 became light pink, but C1 stayed white (compare with restriction analysis 13.6.13)

Preparation of media for inoculation on Sunday (by Katrin Gunka)

• For Plate Reader assay: 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6, negative control DH5α in 4 ml LB
• For Plasmid Mini-Preparation: 10 ml LB + 10 μl Amp for inoculation of C2 Part 2

• Reactions/Master Mixes: see Excel-sheet “dropbox > iGEM > Reporter-Team > digestion system 2hour”
• Plasmids: parts 1 – 6 (plasmids purified on 7.6.13) and part 7, 8 (plasmids purified on 11.6.13)
• 1x reaction with single enzyme:

  Component	Volume
  Enzyme	0.5μl
  Buffer 10x	1μl
  Plasmid DNA	200ng
  Total volume	10μl

  
  For double digestion:
  0.5 μl of each enzyme in case of EcoRI and PstI; for SpeI and PstI, ratio 1:2 is recommended ? 0.5 μl SpeI and 1 μl PstI used
• Incubation at 37°C for1.5 hours
• Additon of 5xDNA-Loading Dye to whole reaction
• Loading of 8 μl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder as marker
• Run at 200 V for 1.5 h in 1xTAE
• EtBr staining ca. 45 min, destaining in water ca. 30 min
• UV detection

Expected Fragments:

Gel:See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

• Partial digestion (next time: longer incubation time, less plasmid…)
• For parts 1, 3, 4, 6, 7 and 8, the expected bands were observed (ok)
• Band pattern of part 5 resembles that expected for parts 1 – 4 -> this plasmid contains RFP gene
• In case of part 2, SpeI was unable to cut the plasmid -> no cleavage for SpeI single digest + linearization for SpeI/PstI double digest -> plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)
• For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short ? think of other cloning strategy without gel extraction (e.g. “play” with resistances of plasmid backbones to digest backbone after cutting out part…)

Preparation of DarR PCR samples for sequencing

4 μl of DarR seq PCR product of reaction 1 + 1 μl of iGEM_34 (fwd) or iGEM_35 (rev)

• kgun_1 -> iGEM_34
• kgun_2 -> iGEM_35

Sequencing at G2L

stored in -70 °C (red box)

• harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation ? stored at – 20 °C in red box
• harvesting of remaining culture for today’s prep
• Performed as on 7.June.13

NanoDrop – Plasmid concentrations

• for DarR seq PCR reactions 1 - 4
• with QIAquick PCR purification Kit (Qiagen), according to quick start manual
• ca. 50 μl of PCR reaction + 250 μl of PB buffer
• reactions 2 and 4 turned violet ? addition of 10 μl NaAc 3.3 M, pH = 5.0
• elution: 30 μl pre-warmed HPLC water directly applied to membrane, incubation for 2 min at RT, centrifugation
• purified PCR products stored at – 20 °C in red box

10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Preparation of 250 ml LB broth media (stored on shelf above bench)

10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

• dilution of primer stocks 1:20 (100 pmol -> 5 pmol):95 μl HPLC water + 5 μl primer 100 pmol
• preparation of dNTP mix -> dilute stocks 1:8 (100 mM ? 12.5 mM):50μl dNTP + 200μl dH₂O
• diluted primers and dNTP mix stored in red box at -20 °C

1x reaction(50μl)

Master Mix for 6 reactions

addition of template (chrom. DNA/dH2O), polymerase and buffer individually, then addition of 37 μl master mix

For tested combinations of buffer and Pol: see table for gel loading

PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)

• Protocol of 1 % Agarose-1xTAE gel
• 4 μl PCR reaction + 1 μl 5x DNA Loading Dye
• 3 μl 1 kb ladder (Quick Load)
• Run at 100 V in 1xTAE buffer
• Staining in EtBr and destaining in water
• UV detection

Loading scheme

Reactions stored at - 20°C in 50 ml Falcon