User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/14

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01/14/11

  • ✓ Assemblies: KAH173, 174
  • ✓ Order primers: for p65 and SP1 activation domain BioBricks



Assemblies
> Re-do KAH173 using dephos vector and max. insert

  1. KAH173: HPK/(E/S)/516 ✓ + KAH73/(E/X)/~5638 ✓
  2. KAH174: KAH75/(E/S)/558 ✓ + (1) KAH57/(E/X)/~4626


> Digests (Fermentas FD)

Reagent Volume   1/14/11 cloning digest
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. KAH57 (E/X) 0.506 1.81 25.3


> Dephosphorylation (Roche)

  1. KAH73(E/X)
Reagent Volume
DNA (clean digest) 10.7 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 6.3
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations (1/15/11)

Ligation Plate results (lig : neg crtl) 1/16/11
1. HPK(E/S)/516, 4.6 ng + KAH73(E/X)/~5638, 25 ng KAH173 >10:1 (Pick 2)
2. KAH73(E/X)/ 25 ng  
3. KAH75(E/S)/558, 6.0 ng + KAH57(E/X)/~4626, 25 ng KAH174 >10:1 (Pick 2)
4. KAH73(E/X)/ 25 ng  
  1 2 3 4
Insert DNA 3.0 --- 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O --- 3.0 2.0 3.0
  10 μL 10 μL 10 μL 10 μL

--> 10 min./ R.T.; add to 30 μL DH5α turbo



New BioBricks: p65, SP1 activation domains
--> Will PCR-clone these from human genomic DNA

> p65

  • a.k.a. NFκB p65 subunit, RELA
  • 261 amino acids from C-terminus (excluding stop codon) (Liu et al., 2001)
  • Contains two adjacent PstI sites
  • Primers:
  1. BB_p65 f1: 5'-CCTTTCTAGAGGAGCTGATCTGACT
  2. BB_p65 r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGATACAGACGATCGT
  3. mut_p65 1: 5'-ATCAAACTGCAaCTGCAaCAGGGCCTCTGA

--> Note (1/21/11): wrong ref sequence; redesign primers!


> SP1

  • Has two activation domains that are sufficient for transcription at TATA/Inr promoters
  • SP1AB is unable to activate a TATA-only promoter; will be interesting to see how this impacts global expression
    • SP1A: aa's 83-262, 180 aa, 540 bp; 1 PstI site
    • SP1B: aa's 263-542, 280 aa, 840 bp; 2 PstI sites
    • SP1AB: aa's 83-621, 539 aa, 1617 bp; 3 PstI sites (clone & mutate this first, then use as template for A and B)
  • Primers
  1. BB_SP1A f1: 5'-CCTTTCTAGAGGCCCGAGTCAGTCA
  2. BB_SP1A r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTCCCATTCAGGGCCAC
  3. mut_SP1A 1: 5'-AACAGCTGCAaTTTGCTGCC
  4. BB_SP1B f1: 5'-CCTTTCTAGAAACATCACCTTGCTA
  5. BB_SP1B r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGATTCCTGAAGTACC
  6. mut_SP1B 1: 5'-CGTTTCTGCAaCTACCTTGA
  7. mut_SP1B 2: 5'-TACAGCTGCAaAACCTCCAA
  8. BB_SP1AB r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGCCAGGATCCCCCGA



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