Team:Goettingen/NoteBook w11

Gel and Nanodrop measurements of R.D. from friday (Jonathan)

Gel:

M|CFP|CFPr.d.|YFP|YFPr.d.|Part1|Part1r.d.|Part2|Part2r.d.|Part3|Part3r.d.|Part4|Part4r.d.|RiboA|RiboAr.d.

M|RiboD|RiboDr.d.|RiboC|RiboCr.d.

àRiboB was forgotten

Nanodrop:

New inoculation of part 8 in Lb Amp

Inocculation of GFP clones (part 6.2 clones 1-7)

for cryostocks and a masterplate, which was forgotten previously

Terminatorrev PCR – Gel ex

 -> Terminatorrev PCR from 7.8.13 looks a bit strange (band in neg. control at height of expected PCR product); it could be the primers, however, since the plasmid used as a template is still present in the sample, PCR clean up (as it was done) might not be enough to purifiy PCR product from template. Thus, gel extraction needs to be performed.

- Addition of <7.5 μl to each of the purified samples

- Loading of entire samples on 1 %-agarose-1xTAE-gel

- Loading of 2 log ladder (3 μl)

- Loading of part 7 C1 plasmid as a control (1 μl of 120.5 ng/μl + 1 µl 5xLD + 3 µl water)

- Run at 85 V

 - Gel extraction + purification using Qiagen PCR clean-up/gel extraction kit

- since Terminatorrev PCR product is ca. 190 bp, 1 volume isopropanol was added after the gel pieces were dissolved

- 2 columns used (loading of sample amounts corresponding to max. 400 mg gel in total per column); when loading one column, I pipetted some of the sample onto the TARA-tube by mistake… so this part of the sample was lost…

- elution from both columns with 30 μl HPLC water each into the same tube

- concentration (NanoDrop):

Terminatorrev PCR – Repeated

 - since PCR from 7.8.13 had a strange negative control, the PCR was repeated as on 7.8.13, but with some modifications:

- with only 2 μl primers (iGEM_71/72)

- 5 reactions in total:

a) neg. control 1: no primers --> which bands are caused by primers?

b) neg. control 2: no template --> which bands are caused by template?

c - e) 3 reactions with template and primers

- template: part 7 C1 plasmid, dilution from 7.8.13 used

- 6x MasterMix containing PfuS, 5x HF buffer, water and dNTPs

- after PCR: 3 reactions with template and primers pooled into 1 tube

- 1%-agarose-1xTAE gel (from Nina)

- loading of 3 μl 2 log ladder

- loading of 4 μl of each PCR reaction + 1 μl 5xLD

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

- samples ran almost out --> no conclusion possible --> gel run repeated:

- gel run:

- 1%-agarose-1xTAE gel

- loading of 3 μl 2 log ladder

- loading of 4 μl of each PCR reaction + 1 μl 5xLD

- (Loading of Terminatorrev PCR product digest, as well – see below; loading of digest of part 6.2 C1 plasmid with EcoRI, too – see below)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> band observed in neg. control results indeed from primers --> one can work with the old PCR product

-> green PCR tube with pooled PCR reactions stored in 15 ml Falcon tube at – 20°C for purification

Restriction of Part 6.2 C1 plasmid with EcoRI to generate vector for ligation with hybridization oligos of promoters

1500 ng plasmid (6 μl of 261.5 ng/μl sample; purified on 9.8.13)

4 μl EcoRI FD

4 μl 10x FD buffer

26 μl water

in total 40 μl

 - digest for 1 h at 37 °C

- gel run:

- 1 %-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 1 μl plasmid (uncut) + 3 μl water + 1 μl 5x LD

- Loading of 3 μl RD reaction + 1 μl water + 1 μl 5x LD

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> digest is still incomplete

-> addition of 1 μl EcoRI FD, 1 μl 10x FD buffer, 8 μl water

-> digest for another 1 h at 37 °C

- gel run:

- 1%-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 1 μl uncut plasmid + 3 μl water + 1 μl 5x LD

- Loading of 4 μl RD reaction + 1 μl 5x LD

- (Loading of Terminatorrev PCR product digest, as well – see below; repeat of gel run of new Terminatorrev PCR, as well – see above)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> Digest only slightly incomplete --> tolerable --> purify vector for further digest with XbaI with PCR clean-up kit

Purification:

 - Qiagen PCR purification kit

- 600 µl PB buffer used (by mistake…)

- Elution 1x with 30 µl HPLC-water (pre-warmed) with incubation at 50 °C for 2 min

- NanoDrop: concentration

- Samples stored in To-do-Box

Restriction of Terminatorrev PCR product obtained after gel ex with EcoRI and PstI (for ligation with pSB1C3)

30 μl PCR product

3 μl EcoRI FD

3 μl PstI FD

4 μl 10x FD buffer

40 μl in total

- digest for 1 h at 37 °C

- gel run:

- 1%-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 4 μl uncut PCR product + 1 μl 5x LD

- Loading of 4 μl RD reaction + 1 μl 5x LD

- (Loading of part6.2 C1 plasmid EcoRI 2 h digest, as well – see above; repeat of gel run of new Terminatorrev PCR, as well – see above)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> PCR product still present/ no over-digest --> PCR clean-up to purify insert for ligation

Purification: 

- Qiagen PCR purification kit

- 500 µl PB buffer used

- Elution 1x with 30 µl HPLC-water (pre-warmed) with incubation at 50 °C for 2 min

- NanoDrop: concentration

- Samples stored in To-do-Box

DarRrev PCR: Test-PCR for optimal primer concentration 

- DarRrev primers (iGEM_83 and iGEM_84) arrived --> dissolved in HPLC-water and preparation of 1:20 dilution in HPLC water

- iGEM_83 and iGEM_84 tend to form strong secondary structures --> test for optimal primer concentration in PCR

- Test- PCR done as on 24.6.13: 5x MasterMix containing water, 5x HF buffer, PfuS, dNTPs; 2 μl, 4 μl and 8 μl primer tested, neg. control w/o template (M.smegmatis chrom.DNA); Annealing temperature of both primers is the same as for iGEM_33 and iGEM_44, since only the prefix and suffix sequences were switched)

- PCR taken out and stored at 4 °C (small fridge) in yellow-tip-box-rag

Sequencing results of Promoter3op cloning in front of part 6 and into shipping vector 

-> Plasmids part 6.2. C1 and part 6.2. C2 contain the Promoter3op insert at the desired site in the vector w/o any mutations --> part 6.2 C1 used for further cloning

->Plasmid Promoter3op in pSB1C3 C1 (see: Colony PCR from 8.8.13) contains the Promoter3op insert at the desired site in the vector w/o any mutations --> inoculation of C1 in 4 ml LBCm for MiniPrep and CryoStock, incubation ON at 37 °C, ca. 200 – 210 rpm

Ligation of pSB1C3 (E+P-digested) with Terminatorrev insert (E+P digested)

- Ligation calculator: for ratio vector: insert= 1:3 and 50 ng vector (2070 bp), ca. 14 ng insert (190 bp) are required 

-> Ligation reactions incubated ON at 16 °C (cold room)

Preparation of LBCm plates 

- From 500 ml medium

- 35 µg/ml Cm in plates

Ligation of Riboswitch Constructs A-D into pSB1C3(from part 7)

For that, ~45ng Vector were ligated to ~150ng Inserts as follows:

                  1.5µl pSB1C3

                  4.5µl Insert (Ribo A-D)

                  2µl T4 Buffer

                  2µl T4 Ligase

                  10µl H20

         = 20µl reaction

Incubation overnight at 16°C