Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30

Ligation/Amplification of DBN002 & DBN003

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Overview Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. Also going to try conventional assembly using the BioBricks method . Type IIS Digestion/Ligation Reagent Volume (µL) Insert 1 (DBN002) 200 ng (11.9 µL) Insert 2 (DBN003) 500 ng (7.5 µL) 10x FD Buffer (clear) 2.5 10mM DTT 1 10mM ATP 1 FD BsaI 1 T4 DNA ligase 0.5 M.B. H2O 0 Total 25.4 Incubate the ligation reaction in a thermocycler (program name DBN IIS): 37°C 5 min 23°C 5 min Cycle the previous two steps for 6 cycles (total run time 1h) 4°C hold until ready to proceed --Clean-up of IIS Ligation-- Use the Qiagen kit. If possible, final volume elution of 30 µL. --PCR-- Reagent Volume (uL) 2x master mix 25 MB grade H2O 0 DBN003 f1 primer 0.25 DBN002 r1 primer 0.25 template 24.5 Total 50 Step Temperature °C Duration Initial denature 95 120 sec Main Cycle (x40): denature 95 30 sec anneal 59 30 sec extension 72 120 sec final extension 72 300 sec soak 4 indef. BioBricks Cloning Follow the method described here. Parts to be used are KAH57 (AmCyan, AmCyan, NLS, Stop) and KAH104 (HPK, Kozak). Both are in v0120 vector. For this cloning, KAH57 will be the vector and KAH104 the insert, placed in front of the KAH57 part. Enzymes used will be E/X for KAH57 and E/S for KAH104. Reagent Volume (KAH57) Volume (KAH104) DNA 25 15 10x Green FD Buffer 3 3 EcoRI 1 1 SpeI 0 1 XbaI 1 0 Water 0 10 Total 30 30 During gel extraction, remove the vector from KAH57 (should be only large piece in digest) & the insert from KAH104 (540bp). Gel image: Cut out vector (0.134g) and insert (0.140g). Ran a gel extraction using Sigma GenElute Gel Extraction Kit, then measured concentration on the spectrophotometer. Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280 KAH57 5.128 1.458054183 2.2275 KAH104 7.9305 0.907218 2.451 Purity as measured by A260/A280 is poor, and concentration is low. Proceeding with ligation step. 50ng vector * 1/5.1 ng/µL = 10 µL purified KAH57 ng insert = 540bp/4691bp * 2 * 50ng = 11.5 ng, * 1/7.9 ng/µL = 1.5 µL purified KAH104 Reagent Volume (µL) vector 10 insert 1.5 Roche Buffer 2x 10 NEB T4 ligase 2 Total 23.5 Incubate for 10 minutes at room temperature. Transformation Transform DH5α-Turbo with: ✓ KAH57_v0120 ✓ KAH104_v0120 ✓ pMax-GFP (brochure says it needs a Kanamycin plate, but René says she used Amp, so try that first) ✓ ligation product of KAH57 & KAH104 ✓ negative control (water) ✓ negative control (digested KAH57 only)