User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/21

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

01/21/11

  • ✓ Oligos: order sequencing primers for pSB31
  • ✓ PCR cloning: p65 and SP1AB activation domains
  • ✓ Oligos: order new p65 BioBrick oligos
  • ✓ Cultures: pSB31 from rt plate (2); 5 mL each



Oligos
> Use sequence around EcoRI cloning site (from Manching)
> IDT DNA

  1. seq_pSB31 f: 5'-CGTTTAGTGAACCGTCAGATC
  2. seq_pSB31 r: 5'-CATTCTAGTTGTGGTTTGTCC


> p65 (RelA), aa's 288-548/ bp 862-1644; Contains 2 PstI sites

  1. BB_p65 f1: 5'-CCTTTCTAGAtacctgccagataca
  2. BB_p65 r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTctgactcagcagggc
  3. mut_p65 1: 5'-ggccctgctgcaactgcaatttgatgatga



PCR
> First, PCR and clone p65 and SP1AB into V0120 using E/S (w.t. parts contain PstI sites)
> Use U2OS cDNA as template

  1. FTRx no-rot, 7/08/10
  2. n.t. kd 8 dy, 11/08/09

> Primers

  1. BB_p65 f/ r
  2. BB_SP1A f/ BB_SP1AB r
Reagent Volume
cDNA 1.0
10 μM primers 1.0
2x GoTaq mix 25.0
dH2O 23.0
  50.0 μL

> BioRad DNA Engine, block A

  • 95 °C/ 3 min.
  • [95°C/ 30 sec.; 55°C/ 30 sec.; 72°C/ 1 min.] x30
  • 72°C/ 3 min.
  • 4°C/ ∞


> Zymo clean; elute w/ 25 μL dH2O


> Digests (Fermentas FD)
--> X/S digest

Reagent Volume   Expected:
1. FTRx/ p65 = 783
2. nt kd/ p65 = 783
3. FTRx/SP1AB = 1617
3. nt kd/SP1AB = 1617
01/21/11 E/S digest of PCR
30 μL/lane, 1% agarose
PCR 25.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.

--> p65 did not work
--> Note: Double checked p65 sequence. Ref sequence was wrong. Discard old primers and replace with new primers designed from correct human p65 CDS.
--> Proceed w/ SP1AB cloning


> Measure conc.'s

Sample OD260 260/280 ng/μL
2. SP1AB w.t. (X/S) 0.177 2.02 8.9


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. SP1AB w.t.(X/S)/1617, 25 ng + V0120(X/S)/~3200, 25 ng SP1AB w.t./V0120 1:1
2. V0120(X/S)/ 25 ng  
  1 2
Insert DNA 3.0 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 ###
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  10 μL 10 μL


--> ~10 min./ r.t.; Add to 30 μL DH5α turbo
--> Forgot to dephos. the vector! Re-do tomorrow.


Personal tools