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Takara Bio USA, Inc.

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Takara Bio USA, Inc.

Cellartis® iPSC rCas9
Electroporation and
Single-Cell Cloning
System User Manual

Cat. No. 632643
(030619)

Содержание Cellartis iPSC rCas9

Страница 1: ...hnical Support techUS takarabio com United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 565 6999 Page 1 of 24 Takara Bio USA Inc Cellartis iPSC rCas9 El...

Страница 2: ...Overview 9 B Generating the DNA Template 10 C Performing the In Vitro Transcription IVT Reaction 12 D Purifying the Transcribed sgRNA 14 VII Editing hiPS Cells by Electroporation 15 A Protocol Overvie...

Страница 3: ...CR product 12 Figure 7 sgRNA yield over time 13 Figure 8 Workflow for electroporation based delivery of rCas9 sgRNA complexes 15 Figure 9 Recommended density of starting culture used for single cell c...

Страница 4: ...omponents have been delivered successfully into target cells through a variety of approaches including vector based expression systems transfection of RNA and introduction of Cas9 sgRNA ribonucleoprot...

Страница 5: ...elivered Once the rCas9 sgRNA RNP complexes have been delivered by electroporation and cells have recovered single cells must be isolated and expanded into clonal cell lines in order to isolate and sc...

Страница 6: ...PSC rCas9 Electroporation and Single Cell Cloning System for other human iPS cell lines or for Cellartis iPS cells grown in another system please be aware that these cell lines will need to be adapted...

Страница 7: ...r equivalent NanoDrop 2000 spectrophotometer Thermo Fisher Scientific Cat No ND 2000 or equivalent Electroporation Supplies Use of this product requires an electroporator electroporation chamber typic...

Страница 8: ...mended to transfer cells from other culturing systems to the Cellartis DEF CS 500 Culture System Cat No Y30010 before editing and single cell cloning with the Cellartis iPSC rCas9 Electroporation and...

Страница 9: ...cting an appropriate DNA sequence at the target region is critical for maximizing the potential for efficient cleavage at the target site and for minimizing the likelihood of non specific cleavage eve...

Страница 10: ...your sgRNA target sequence and the Guide it Scaffold Template specific sequence Figure 5 Choosing the Correct DNA Target Sequence Choose the DNA target sequence that will correspond to your actual sgR...

Страница 11: ...add extra Gs for transcription initiation Extra Gs could reduce cleavage efficiency c Your specific sgRNA target sequence 20 nt d The Guide it Scaffold Template annealing sequence 15 nt at the 3 end o...

Страница 12: ...ater 11 Total 25 2 Place reactions in a preheated thermal cycler with a heated lid and run the following program 33 cycles 98 C 10 sec 68 C 10 sec 4 C forever 3 Run and analyze 5 l of the PCR product...

Страница 13: ...a 4 hr incubation but a shorter incubation time is acceptable if you do not need to maximize your sgRNA yield see Figure 7 We have observed a clear sharp band via Agilent Bioanalyzer following an 8 h...

Страница 14: ...50 l of IVT Wash Buffer and centrifuge at 11 000g for 2 min at room temperature 9 Place the IVT RNA Clean Up Spin Column in a new 1 5 ml microcentrifuge tube 10 Add 20 l of RNase Free Water directly o...

Страница 15: ...ell recovery step prior to single cell cloning Section VIII Please note that when the following protocol specifies use of COAT 1 it is referring to the DEF CS COAT 1 from the Cellartis DEF CS 500 Cult...

Страница 16: ...n use components of the Cellartis DEF CS 500 Culture System to prepare enough DEF CS medium to 1 neutralize the TrypLE Select Enzyme 1X used to dissociate cells from the initial culture vessel a 1 10...

Страница 17: ...ion 1 Thaw Guide it Recombinant Cas9 Electroporation Ready and sgRNA solutions at room temperature NOTE We recommend preparing aliquots upon initial thawing of Guide it Recombinant Cas9 Electroporatio...

Страница 18: ...1 100 v 20 ms 2 pulses 4 Gently resuspend the cells by tapping and then transfer 7 5 l of the cell suspension into the tube containing the 7 5 l of rCas9 sgRNA solution 5 Mix well by gently pipetting...

Страница 19: ...System for further upscaling Table IV describes a schedule of all media changes volume and composition necessary to create clonal lines in 24 well plates that are ready for culture with the Cellartis...

Страница 20: ...solution per well see Table V for guidance Table V Preparation of coating solution for a 96 well plate Number of wells 96 well plate Volume of diluted coating solution l Volume of SCC COAT 1 l Volume...

Страница 21: ...s necessary 2 Aspirate the medium from the culture vessel and wash the cell layer once with D PBS 3 Add TrypLE Select Enzyme 1X to the culture vessel using the amount indicated in Table VI Place the v...

Страница 22: ...ay Preparing DEF CS SCC Medium for Establishment of Single Cell Colonies 1 Prepare 150 l per well of DEF CS SCC medium by adding DEF CS GF 1 dilute 1 333 GF 2 dilute 1 1 000 and GF 3 dilute 1 1 000 to...

Страница 23: ...idity for a minimum of 3 hr 5 Aspirate the diluted SCC COAT 1 solution from the 48 well plate immediately before use B Preparing DEF CS SCC Medium for Passaging 1 Prepare the appropriate volume of DEF...

Страница 24: ...argeting Nat Biotechnol 32 347 55 2014 For information on using the 4D Nucleofector System to electroporate cells please visit http www lonza com products services bio research transfection genome edi...

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