Team:Heidelberg/Notebook/miMeasure/September

13/09/2010

Restriction digest of psiCHECK-2 plasmid
This will be used as backbone for raPCR cloning. Enzymes: XhoI and NotI

Assay:

5 µL 10x NEBuffer 3

5 µL 10x BSA

5 µL plasmid (psiCHECK-2, ~370 ng/µL)

3 µL XhoI

1 µL NotI

18.6 µL H₂O

Restriction digest was performed for approx. 5h

raPCR to create binding sites for different miRNAs This random assembly PCR (raPCR) will be done to create binding site patterns for the miRNAs mentioned. In the first PCR step the oligos will basically anneal and constructs of different lengths will form. In the second step, the stop oligos are used as primers to amplify the previously formed constructs.

• first tries are: hsa-mir-122, hsa-mir122(ran9-12) and mm-mir-376a/375 (Oligos: ra001-003 and ra006)
  

stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI (ra017/018)
spacer: raPCR_AS13-spacer(0) and raPCR_AS13-spacer(10) (ra012/013)

Oligos were used in standard conc. (100µM)

• 1. PCR

Total: 12 reactions
each reaction was set up in 30 µL, using 2x Phusion Mastermix for 12 cycles

• PCR purification: each PCR was purified using Qiagen PCR purification Kit and eluted in 32 µL
  

for the next PCR, three assay will tried:

1. 5µL eluate + 1 µL of each stop oligo in 50µL total volume
2. 5µL eluate + 2 µL of each stop oligo in 50µL total volume
3. 20µL eluate + 1 µL of each stop oligo in 50µL total volume
   
   

stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI

• 2. PCR

In total there were 72 reactions. Each was run with 2x Phusion Mastermix, missing volume was filled with water.

DNA was stored in fridge afterwards

14/09/2010

72 PCRs were analyesed on 1% agarose gel

Best looking lanes of spacer(0) and spacer(10) were choosen for preparative gel

preparative gel (1.5%) was made for miR122

Sample for small Binding site patterns (100-250 bp) and large patterns (250-400 bp) were taken.

each were eluted in 1 column (a lot of work for gel extraction...)

15/09/2010

Gel extracted samples were digested with Not/Xho for cloning into psiCheck-2 vector: 5µL template in total 30µL using 1µL xho and 0.6µL Not enzyme

digest was then purified using qiagen nucleotide removal kit, elution in 30µL

due to a problem of the vector-size (it has 8000 instead of 6000 bp), ligation was set-up overnight at roomtemperature

ligation: 2µL buffer 1µL ligase 7µL water 1µL backbone (6000bp) 9µL restriction digest

16/09/2010

Transformation of ligations: 5µL ligation assay in 50µL TOP10 E.coli 25 min on ice 45sec heat shock on 42°C 1.5-2h shaking at 37°C

plated 200µL on Amp-Plates

after incubating ~8h, at 37°C, the plates were incubated overnight at room temperature

17/09/2010

Colonies were visible in reasonable numbers on every plate

colony pcr was performed to check clones

20µL assay PCR assay, using 2x PCR master mix (Taq) from Fermentas

one colony was dissolved in 20µL water

5µL of this bacteria solution was used as PCR template

PCR conditions as recommended from Fermentas

Minipreps will be setup in the evening for overnight cultures (4mL, Amp)

5 for each construct

28/09/2010

• send 1.3, 1.5, 1.7, 3.8, 3.2 for sequencing

29/09/2010

• got sequencing results of 1.3, 1.5, 1.7, 2.8, 3.2.

• 1.3 - 3x - all ok
• 1.5 - 2x - both ok
• 1.7 - 3x - all ok
• 2.8 - not ok
• 3.7 - 2x - both ok seperated by 10 nucleotide spacer

30/09/2010

• raPCRfrom above(1, 2, 3, 4)
• PCR purification ( nanodrop: c ≈ 100ng/µl)
• digested: 2 x 1 µg DNA:

• EcoRI
• PstI

• gel purification (nanodrop: c ≈ 25ng/µl)
• Ligation (Quick Ligase and overnight ligation with T4 ligase) into pSB1C3

• Vector ≈ 2000 bp
• Insert ≈ 200 bp

• Transformation