# Cloning and sequencing ## expand\_more ## Prepared by Jian-Quan Ni, Matt Booker, Norbert Perrimon\* \*Questions? Contact Jian-Quan Ni at . VALIUM10 cloning VALIUM20 and VALIUM22 cloning Sequencing inserts ## VALIUM10 cloning **For VALIUM10 (or WALIUM10)** **Click [here](/file_url/165) to download pdf** **1. Oligo design** The TRiP oligos were designed using [SnapDragon](/snapdragon) with the following criteria: 1. Product size was set to 400 to 600 bp 2. 19 bp predicted off-targets were avoided 3. Only splice-form universal portions of each gene were selected for oligo design **2. PCR** SortForward primer 20uM 0.5ul Reverse primer 20uM 0.5ul DNA Template (make sure to use genomic DNA or cDNA) 1ul Taq polymerase mix (Promega, Cat No. M7122) 12.5ul Add ddH2O to 25ul total volume Once the PCR finishes (30 Cycles), load 10ul onto an agarose gel to verify the quality and quantity of the PCR product. If multiple bands are seen, cut out the relevant band and purify it. **3. TOPO Ligation** SortPCR product (there’s no need for purification) 1ul pENTR/D-TOPO entry vector (Invitrogen, Cat.No. K240020) 1ul Salt Solution (1.2M NaCl, 0.06M MgCl2) 1ul Add ddH2O to 6ul total volume Mix, incubate at room temperature for 10 min **4. Transformation** Transfer all ligation mix to 50ul TOP10 cells, place on ice for 30 min, heat shock, add SOC medium and incubate at 37°C for 50 min, then plate (Kan resistant). **5. Select clone, culture in LB medium (Kan resistant) and miniprep.** **6. Sequence pENTR/D-TOPO entry vector to ensure the insertion and its orientation is correct; i.e., 3’ to 5’.** Oligo used for sequencing, M13-F: 5’-TTGTAAAACGACGGCCAGTC-3’ **7. Recombination** Sort45-85ng/ul pENTR/D-TOPO entry vector (from step 5) 0.3ul 200ng/ul VALIUM10 (or WALIUM10) 0.3ul LR clonase (Invitrogen, Cat No. 11791-100) 0.8ul Add ddH2O (or TE buffer, pH8.0) 4ul total volume Mix, incubate at 25°C for 1 hr, and then add 1ul proteinase K (From LR clonase kit), further incubate at 37°C for 10 min to remove the recombinase. **8.Transformation** SortRecombination product 5ul TOP10 competent cells 50ul Mix, incubate on ice 30 min, 42°C heat shock, add SOC medium, incubate at 37°C for 30 min, then plate (Amp resistant) **9. Select clone, culture in 5ml LB medium (Amp resistant) and miniprep** At end elute with 100ul ddH2O. **10. Sequence hairpin construct to confirm the ftz intron is in the correct orientation** (in our experience: 40-60% of the clones are correct). Primer used for DNA sequencing, Hsp70F: 5'-CGCAGCTGAACAAGCTAAAC-3' **11. Precipitation (DNA from step 9)** SortDNA 90ul 3M NaAc, pH5.2 10ul EtOH 500ul Mix, -20°C for 1 hr **12. Centrifuge (15,000 rpm, 20 min), wash once with 70% EtOH, and dissolve in 40ul ddH2O** **13. Construct is ready for injection** **Note:** The only difference between VALIUM10 and WALIUM10 is their selectable eye color marker; VALIUM10 uses Vermilion, WALIUM10 uses White. ## VALIUM20 and VALIUM22 cloning **For VALIUM20 and VALIUM22** **Click [here](/file_url/166) to download pdf** **1. Oligo design:** Select a 21nucleotide sequence based on the algorithm of Vert et al. (2006). This oligo design eliminates off target effects starting at 16 nucleotides. Based on a miR1 scaffold, for the top strand oligo, add ctagcagt to the 5’ end of the passenger strand DNA, add tagttatattcaagcata between the passenger strand DNA and the guide strand DNA, add gcg to the 3’ end of guide strand DNA, so the resulting oligo will be: ctagcagt**NNNNNNNNNNNNNNNNNNNNN**tagttatattcaagcata**NNNNNNNNNNNNNNNNNNNNN**gcg For the bottom strand oligo, add aattcgc to the 5’ end of the passenger strand DNA, add tatgcttgaatataacta between the passenger strand DNA and the guide strand DNA, add actg to the 3’ end of the guide strand DNA, so the resulting oligo will be: aattcgc**NNNNNNNNNNNNNNNNNNNNN**tatgcttgaatataacta**NNNNNNNNNNNNNNNNNNNN**actg **2. Annealing the top and bottom strand oligos:** Add 10ul top strand oligo (10-20uM) and 10ul bottom strand oligo (10-20uM) into 80ul annealing buffer (10mM Tris-HCl, pH 7.5, 0.1M NaCl, 1mM EDTA). Mix, incubate at 95ºC for 5 min, then slowly cool down to room temperature. The resulting DNA fragment has overhangs for NheI and EcoRI. **3. Ligation:** Directly clone this DNA fragment into a VALIUM20 or VALIUM22 vector, which has been linearized by NheI and EcoRI. 6ul annealing product 2ul 10X ligation buffer 1ul T4 DNA ligase (1U/ul) 1ul 40ng/ul backbone (gel purified VALIUM20 or VALIUM22 cut with NheI and EcoRI) Add ddH2O to 20ul total volume. Mix, incubate at 16oC for 1 hour. **4. Transformation:** Add 10ul ligation product into 50ul TOP10 competent cells, following standard transformation protocol. **5. Colony selection:** PCR select the correct clone by appearance of a 350bp PCR product using the following PCR primers: SortpVALIUM20: F: 5’-ACCAGCAACCAAGTAAATCAAC-3’ R: 5’-TAATCGTGTGTGATGCCTACC-3’ pVALIUM22: F: 5’-GGTGATAGAGCCTGAACCAG-3’ R: 5’-TAATCGTGTGTGATGCCTACC-3’ **6. Sequencing:** Confirm the correct shRNA construct using the following sequencing primers: pVALIUM20: 5’-ACCAGCAACCAAGTAAATCAAC-3’ pVALIUM22: 5’-GGTGATAGAGCCTGAACCAG-3’ **7. DNA miniprep and injection.** ## Sequencing inserts **Sequencing TRiP lines to test for the presence of a VALIUM insertion** For VALIUM1, the primers are: F: 5'-CGCAGCTGAACAAGCTAAAC-3' R: 5'-CGACTGCGAATAGAAACTCAC-3' For VALIUM10, the primers are: F: 5'-CGCAGCTGAACAAGCTAAAC-3' R: 5'-CTAGACTGGTACCCTCGAATC-3' For VALIUM20 and VALIUM21 the primers are: F: 5'-CGCAGCTGAACAAGCTAAAC-3' R: 5’-TAATCGTGTGTGATGCCTACC-3’ For VALIUM22 the primers are: F: 5’-GGTGATAGAGCCTGAACCAG-3’ R: 5’-AATCGTGTGTGATGCCTACC-3’