User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/23

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04/23/10

  • ✓ Minipreps: KAH154-159/MV2
  • ✓ Sequencing: KAH156, 157/MV2



Minipreps
> Check with XbaI digests

Reagent Volume Expected:
1. KAH154/MV2 = 1724
2. KAH155/MV2 = 1724
3. KAH156/MV2 = 1724
4. KAH157/MV2 = 1724
5. KAH158/MV2 = 2688
6. KAH159/MV2 = 2691
File:KAH 042310 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
XbaI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

Sequencing (Genewiz, standard)
--> Can't sequence old KAH156/MV2, used up last of it in transfection
--> Need to confirm 156, 157 in order to have proper id for transfected cells

  1. KAH156/MV2 (new prep) DDB122: fshPcCD! (should be flyPcCD)
  2. KAH156/MV2 (new prep) DDB123: fshPcCD! (should be flyPcCD)
  3. KAH157/MV2 (new prep) DDB122: flyPcCD! (should be fshPcCD)
  4. KAH157/MV2 (new prep) DDB123: flyPcCD! (should be fshPcCD)
  5. KAH157/MV2 (old prep) DDB122: bad seq
  6. KAH157/MV2 (old prep) DDB123: flyPcCD! (should be fshPcCD)


--> Looks like fly and fish PcCD's were mixed up during cloning. Take note of this and continue. May want to check other transfection constructs (including VP64+ set) just to be sure the id's of the stable lines are correct.