Haynes Lab:Notebook:Shared/Cloning Bootcamp/2015/11/20

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Baylee Murphy- 11/20/15 Session 3

KAH126- MV2 Culture inoculation results from Session 2

  • Liquid cultures 1 and 2 both had cloudy yellow broth, indicating growth
  • Streaks for both showed colony growth

Samples for mini prep and analysis

  • (1) Sample 1- KAH126-MV2 colony 1, [BB#}; part = [length in bp]; vector= [length in bp]
  • (2) Sample 2- KAH126-MV2 colony 2, [BB#}; part = [length in bp]; vector= [length in bp]

DNA concentration Data

Sample Read# Location 260 Raw 280 Raw 320 Raw 260 280 260/280 ng/µL
BM KAH126-1 C2 0.32 0.207 0.08 0.249 0.131 1.909 249.344
BM KAH126-1 C3 0.313 0.201 0.074 0.245 0.128 1.911 244.882
BM KAH126-2 D2 0.323 0.203 0.06 0.272 0.146 1.861 272.416
BM KAH126-2 D3 0.319 0.2 0.066 0.258 0.136 1.904 258.244



Plasmid Validation

Restriction Digest Table

  • Checked plasmid minipreps with Xba digest
Reagent Volume Expected:
1. KAH126-MV2 = 1934 bp and 4505 bp
Today's gel
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 1.0 μL
10X buffer 1.5 μL
Xba 1.0 μL
dH2O 10.5 μL
  10 μL --> 37°C/ 20 min.

Control for 1 and 2

Reagent Volume
DNA(plasmid) 1.0 μL
10X buffer 1.5 μL
dH2O 11.5 μL


Results of restriction digest show expected bands and indicate Xba enzyme worked

KAH126 incluswa Hpdc, scar, mcherry: about 1000 bp

  • forward primer- DD122 10 uM
  • reverse primer-DD123 10 uM


DNA Sequencing Samples

  • Submitted to DNASU on: '11/20/15'
  • Order Number:
  1. Sample 1- KAH126-MV2 colony 1, forward primer DD122
  2. Sample 2- KAH126-MV2 colony 1, reverse primer DD123
  3. Sample 3- KAH126-MV2 colony 2, forward primer DD122
  4. Sample 4- KAH126-MV2 colony 2, reverse primer DD123