User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/15

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07/15/11

  • ✓ Cultures/minipreps/sequencing order: p65 biobrick (4)
  • ✓ Assemblies: KAH219-224



Minipreps, sequencing order
> Check via sequencing
> Primers: P0001 = f, P0002 = r
> Results: Very poor quality sequences (low peak heights). Use BLAST to find match to p65

  1. KAH017v2-1, f: nothing
  2. KAH017v2-1, r: nothing
  3. KAH017v2-2, f: nothing
  4. KAH017v2-2, r: nothing
  5. KAH017v2-3, f: 92% match (+/+) to p65 1-746 bp
  6. KAH017v2-3, r: 83% match (+/-) to p65 778-1 bp
  7. KAH017v2-4, f: nothing
  8. KAH017v2-4, r: nothing

> Rushed the cultures/minipreps. Try getting better yeild of DNA from streaked clones. Re-do culture and miniprep for clone #3.



Assemblies

  1. KAH219: Kozak/(S/P)/18 ✓ + KAH210/(X/P)/2398 ✓
  2. KAH220: " + KAH211/(X/P)/1321 ✓
  3. KAH221: " + KAH212/(X/P)/1621 ✓
  4. KAH222: " + KAH216/(X/P)/2590 (1)
  5. KAH223: " + KAH217/(X/P)/1513 (2)
  6. KAH224: " + KAH218/(X/P)/1813 (3)


> Digests (Fermentas FD)

Reagent Volume   File:KAH 071511 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH216 (X/P) 0.147 2.39 7.4
2. KAH217 (X/P) 0.160 2.66 8.0
3. KAH218 (X/P) 0.175 3.21 8.8


> Ligations

Ligation Plate results (lig : neg crtl) 07/16/11
1. KAH210(X/P)/2398, 37 ng + Kozak(S/P)/~3218, 25 ng KAH219 >10:1 (Pick 2)
2. KAH211(X/P)/1321, 21 ng + " KAH220 >10:1 (Pick 2)
3. KAH212(X/P)/1621, 25 ng + " KAH221 >10:1 (Pick 2)
4. KAH216(X/P)/2590, 40 ng + " KAH222 >10:1 (Pick 2)
5. KAH217(X/P)/1513, 24 ng + " KAH223 >10:1 (Pick 2)
6. KAH218(X/P)/1813, 28 ng + " KAH224 >10:1 (Pick 2)
7. Kozak(S/P)/ 25 ng  
  1 2 3 4 5 6 7
Insert DNA 5.1 1.5 1.5 5.0 3.0 3.0 ---
Vector DNA 0.4 0.4 0.4 0.4 0.4 0.4 0.4
2x lgn buf (Roche) 6.5 6.5 6.5 6.5 6.5 6.5 6.5
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- 3.6 3.6 --- 2.0 2.0 5.1
  13.0 μL 13.0 μL 13.0 μL 13.0 μL 13.0 μL 13.0 μL 13.0 μL

--> Add 30 μL DH5α Turbo; plate on 100 μg/mL amp