User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/03

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08/03/10 ✓ Assemblies: Pc-ATF's and Pc-RFP's + Kozak ✓ Minipreps: KFV1 (2) Minipreps > Check with separate NotI and SpeI digests Reagent Volume Expected: 1, 3. KFV1/NotI = ~7000(+~35) 2, 4. KFV1/SpeI = ~7000(+~35) 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 enzyme 2.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. --> Keep clone #1; figure out what primer to use for sequencing confirmation (Ian's Actin 5C sequence data) Assemblies KAH160: Kozak(K0001)/(S/P)/~3200 ✓ + (1) KAH109/(X/P)/1123 KAH161: " + (2) KAH111/(X/P)/1123 KAH162: " + (3) KAH112/(X/P)/1123 KAH163: " + (4) KAH113/(X/P)/2107 KAH164: " + (5) KAH114/(X/P)/2110 KAH165: " + (6) KAH148/(X/P)/967 KAH166: " + (7) KAH150/(X/P)/967 KAH167: " + (8) KAH151/(X/P)/967 KAH168: " + (9) KAH152/(X/P)/1931 KAH169: " + (10) KAH1153/(X/P)/1934 KAH170: " + (11) KAH115/(X/P)/931 > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 15.0 10x buffer 3.0 XbaI 1.0 PstI 1.0 dH2O 10.0   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH109 (X/P) 1.057 2.43 52.8 2. KAH111 (X/P) 0.827 2.21 41.4 3. KAH112 (X/P) 1.089 2.53 54.4 4. KAH113 (X/P) 1.380 1.94 69.0 5. KAH114 (X/P) 1.022 2.20 51.1 6. KAH148 (X/P) 0.793 2.25 39.6 7. KAH150 (X/P) 0.660 2.09 33.0 8. KAH151 (X/P) 0.631 2.08 31.6 9. KAH152 (X/P) 1.030 2.12 51.5 10. KAH153 (X/P) 0.980 2.14 49.0 11. KAH115 (X/P) 0.628 2.21 31.4 > Ligations Ligation Plate results (lig : neg crtl) 08/04/10 1. KAH109(X/P)/1123, 18 ng + K0001(S/P)/3200, 25 ng KAH160 >10:1 (Pick 2) 2. KAH111(X/P)/1123, 18 ng + " KAH161 >10:1 (Pick 2) 3. KAH112(X/P)/1123, 18 ng + " KAH162 >10:1 (Pick 2) 4. KAH113(X/P)/2107, 33 ng + " KAH163 >10:1 (Pick 2) 5. KAH114(X/P)/2110, 33 ng + " KAH164 >10:1 (Pick 2) 6. KAH148(X/P)/967, 15 ng + " KAH165 >10:1 (Pick 2) 7. KAH150(X/P)/967, 15 ng + " KAH166 >10:1 (Pick 2) 8. KAH151(X/P)/967, 15 ng + " KAH167 >10:1 (Pick 2) 9. KAH152(X/P)/1931, 30 ng + " KAH168 >10:1 (Pick 2) 10. KAH153(X/P)/1934, 30 ng + " KAH169 >10:1 (Pick 2) 11. KAH115(X/P)/931, 15 ng + " KAH170 >10:1 (Pick 2) 12. K0001(S/P)/ 25 ng 0 colonies   1-11 12 Insert DNA 1.0 --- Vector DNA 0.4 0.4 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O 2.6 2.6   10μL 10μL