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

Test restriction of plasmids containing parts 1 – 8

-          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	0.5 µl
Buffer 10x	1 µl
Plasmid DNA	200 ng
Total volume	10 µl

è     For double digestion:

           0.5 µl of each enzyme in case of EcoRI and PstI;

          for SpeI and PstI, ratio 1:2 is recommended à 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:

Part no.	Fragments for PstI and SpeI (bp)	Plasmid size (linearization with PstI or SpeI)(bp)
1	~900 + 2000	2948
2
3
4
Part no.	Fragments for PstI and EcoRI(bp)	Plasmid size (linearization with PstI or EcoRI)(bp)
5	??? (if RFP gene à same pattern as expected for parts 1 – 4; if no RPF gene à same pattern as expected for part 8)
6	917 2029	2946
7	140 2050	2199
8	53 2038	2091

 

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

Conclusions:

è     Partial digestion (next time: longer incubation time, less plasmid…)

è     For parts 1, 3, 4, 6, 7 and 8, the expected bands were observed (ok)

è     Band pattern of part 5 resembles that expected for parts 1 – 4 à this plasmid contains RFP gene

è     In case of part 2, SpeI was unable to cut the plasmid à no cleavage for SpeI single digest + linearization for SpeI/PstI double digest à plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)

è     For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short à 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)

Tubes:

kgun_1 à iGEM_34

kgun_2 à iGEM_35

-          Sequencing at G2L

Cryo-Stocks of E. coli transfomants (8, 7 C1)

Stored in -70 °C (red box)

 

Plasmid Mini-Preparation of parts 8, 7 C1

-          harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation à stored at – 20 °C in red box

-          harvesting of remaining culture for today’s prep

è     Performed as on 7.6.13

NanoDrop – Plasmid concentrations

Part no.	c(DNA) [ng/µl]	A260/A280	A260/A230
7	67.0	1.98	2.25
8	62.7	1.89	2.08

 

DarR seq PCR purification

-          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 à 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

Colonies on the plates: parts 8, 7 C1

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

backup plates: C1, C2, C3 for part 8

 

Colonies on the plates: parts 8, 7 C1

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

Re-streak of E.coli clones from prtas 6 and 7 on new LB^Chloram plates

Preparation of new LB^Chloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)

 

PCR with DarR primers with different enzymes and different buffers

-          dilution of primer stocks 1:20 (100 pmol à 5 pmol):

95 µl HPLC water + 5 µl primer 100 pmol

-          preparation of dNTP mix à dilute stocks 1:8 (100 mM à 12.5 mM):

50 µl of dATP 100 mM

+ 50 µl of dGTP 100 mM

+ 50 µl of dTTP 100 mM

+ 50 µl of dCTP 100 mM

+ 200 µl dH₂O

è     diluted primers and dNTP mix stored in red box at -20 °C

 

1x reaction (50 µl)

Component	Volume
Buffer (5x GC buffer or 5x HF buffer)	10 µl
dNTP mix (12.5 mM each)	2 µl
Primer fwd iGEM_34 (5 pmol)	2 µl
Primer rev iGEM_35 (5 pmol)	2 µl
Chromosomal DNA M. smegmatis	2 µl
DNA-Polymerase (Phusion or PhuS) or dH2O for water control	1 µl
dH2O	31 µl

 

-          preparation of master mix for 6 reactions containing

Component	Volume
dNTP mix (12.5 mM each)	12 µl
Primer fwd iGEM_34 (5 pmol)	12 µl
Primer rev iGEM_35 (5 pmol)	12 µl
dH2O	186 µl

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

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

 

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

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	60 °C (TA = TM (≈66 °C) – 6 °C)	35 s
Elongation	72 °C	2 min (Phu  needs more time than Phusion!)
Final elongation	72 °C	10 min
Hold	15 °C	∞

-          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

M	1	2	3	4	5	M
1 kb ladder (QuickLoad)	HF	GC	HF	GC	Water controlHF	1 kb ladder (QuickLoad)
	Phusion		PhuS

è     Reactions stored at - 20°C in 50 ml Falcon