User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/02

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08/02/11

  • ✓ Cultures/ minipreps: KAH231, 232, 233 (2 each); KAH213, 219/MV1 (3 each); KAH219, 223, 224/pSB31 (4 each)
  • ✓ Order oligos: for PCR cloning of BB constructs into pSB31



Minipreps
1-6. KAH231-233: check with E/P
7-12. KAH213, 219/MV1: check with E/P
13-24. KAH219, 223, 224/pSB31: check with KpnI/ NcoI

Reagent Volume Expected:
1,2. KAH231 = 1588
3,4. KAH232 = 1780
5,6. KAH233 = 832

Vector frag. = 4072
Insert + 1326...
7-9. KAH213/MV1 = 4160
10-12. KAH219/MV1 = 3748

pSB31 (empty) = 4500, 450
*pSB31 bands
**last frag + 300
13-16. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532**
17-20. 223/pSB31 5-8 = 4500*, 150*, 636, 1190**
21-24. 224/pSB31 5-8 = 4500*, 150*, 636, 1490**

25,26. Kozak retransformation = ~3200, 18

File:KAH 080211 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Pick more colonies for KAH213/MV1, KAH219/pSB31, KAH223/pSB31



Oligo order: IDT
> Blunt cloning and adapter-mediated cloning don't work well. PCR amplify BB construct to have EcoRI at both ends

  1. Eco insert f1: 5'-ccttgaattcgcggccgcttctaga (full BB prefix with 4 bp extra at 5')
  2. Eco insert r1: 5'-ccttgaattctctgcagcggccgctactagt (full BB suffix with EcoRI at 5' plus 4 bp extra)