User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/27

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01/27/11

  • ✓ Assemblies: p65 w.t. (PCR); miR sensors
  • ✓ Sequencing order: Pc-TF/pSB31 clones; SP1AB mut clones



Assemblies
--> Include set from 01/25/11

  1. p65 w.t.: p65 w.t./(X/S PCR)/783 + V0120/(X/S dp)/~3200 ✓
  2. KAH179/176fwd/MV8: KAH179/(E/S)/2941 ✓ + KAH176/MV8/(E/X)/7785✓
  3. KAH180/176fwd/MV8: KAH180/(E/S)/2743 ✓ + "
  4. KAH179/177fwd/MV8: KAH179/(E/S)/2941 + KAH177/MV8/(E/X)/7978 ✓
  5. KAH180/177fwd/MV8: KAH180/(E/S)/2743 + "
  6. KAH179/178fwd/MV8: KAH179/(E/S)/2941 + KAH178/MV8/(E/X)/7989 ✓
  7. KAH180/178fwd/MV8: KAH180/(E/S)/2743 + "


> PCR
--> p65 from cDNA templates (Note: p65 up-regulated in stressed cells; try cDNA from rotenone-stressed cells)

  1. FTRx no rot., 7/08/10
  2. FTRx + rot., 7/08/10
Reagent Volume
cDNA (plasmid) 1.0
10 μM primers 1.0
2x GoTaq 25.0
dH2O 23.0

--> 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 PCR; elute w/ 25 μL dH2O


> Digests (Fermentas FD)

Reagent Volume   PCR digest 01/27/11
30 μL/lane, 1% agarose
Expected = 783 bp
Cut out and purify both bands together
PCR DNA 25.0
10x buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. p65 w.t. (X/S) 0.071 2.28 3.5


> Ligations

Ligation Plate results (lig : neg crtl) 01/28/11
1. p65(X/S)/783, 12 ng + V0120(X/S dp)/~3200, 25 ng p65 w.t. 10:1 (Pick 4)
2. V0120(X/S dp)/ 25 ng  
3. KAH179(E/S)/2941, 26 ng + KAH176fwd/MV8(E/X)/7785, 35 ng KAH179/176fwd/MV8 10:1 (Pick 2)
4. KAH180(E/S)/2743, 25 ng + " KAH180/176fwd/MV8 10:1 (Pick 2)
5. KAH176fwd/MV8(E/X)/ 35 ng  
6. KAH179(E/S)/2941, 26 ng + KAH177fwd/MV8(E/X)/7978, 35 ng KAH179/177fwd/MV8 10:1 (Pick 2)
7. KAH180(E/S)/2743, 25 ng + " KAH180/177fwd/MV8 10:1 (Pick 2)
8. KAH177fwd/MV8(E/X)/ 35 ng  
9. KAH179(E/S)/2941, 26 ng + KAH178fwd/MV8(E/X)/7989, 35 ng KAH179/178fwd/MV8 10:1 (Pick 2)
10. KAH180(E/S)/2743, 25 ng + " KAH180/178fwd/MV8 10:1 (Pick 2)
11. KAH178fwd/MV8(E/X)/ 35 ng  
  1 2 3 4 5 6 7 8 9 10 11
Insert DNA 3.5 --- 3.0 4.5 --- 3.0 4.5 --- 3.0 4.5 ---
Vector DNA 1.0 1.0 1.7 1.7 1.7 1.6 1.6 1.6 4.2 4.2 4.2
2x lgn buf (Roche) 5.5 5.5 7.2 7.2 7.2 7.1 7.1 7.1 9.7 9.7 9.7
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- 3.5 1.5 --- 4.5 1.5 --- 4.5 1.5 --- 4.5
  11 μL 11 μL 14.4 μL 14.4 μL 14.4 μL 14.2 μL 14.2 μL 19.4 μL 19.4 μL 19.4 μL 19.4 μL

--> R.T./ ~15 min.; Add to 50 μL DH5α turbo



Sequencing Order (Genewiz)
--> Results: 1/28/11

  1. KAH160/pSB31-9, seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
  2. KAH160/pSB31-9, seq_pSB31 rev: failed (non-specific)
  3. KAH165/pSB31-1 (#7), seq_pSB31 fwd: insert in reverse orientation
  4. KAH165/pSB31-1 (#7), seq_pSB31 rev: failed (ns)
  5. KAH165/pSB31-4 (#10), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
  6. KAH165/pSB31-4 (#10), seq_pSB31 rev: failed (ns)
  7. KAH170/pSB31-3 (#2), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
  8. KAH170/pSB31-3 (#2), seq_pSB31 rev: failed (ns)
  9. KAH170/pSB31-5, seq_pSB31 fwd: insert in reverse orientation
  10. KAH170/pSB31-5, seq_pSB31 rev: failed (ns)
  11. SP1AB 2/3mut-1, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2 (keep this clone)
  12. SP1AB 2/3mut-1, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
  13. SP1AB 2/3mut-2, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
  14. SP1AB 2/3mut-2, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
  15. SP1AB 2/3mut-3, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
  16. SP1AB 2/3mut-3, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
  17. SP1AB 2/3mut-4, P0001: n/d
  18. SP1AB 2/3mut-4, P0002: n/d
  19. SP1AB 2/3mut-5, P0001: n/d
  20. SP1AB 2/3mut-5, P0002: n/d
  21. SP1AB 2/3mut-6, P0001: n/d
  22. SP1AB 2/3mut-6, P0002: n/d

--> Pc-TF/pSB31: Success! Got all three clones for ES cell transfection experiment. Maxi prep and send to Manching. Reverse primer failed (discard/ design new primer).
--> SP1AB mutagenesis: U2OS seems to have two missense mutations in the SP1AB activation domain (bp 230: Gln -> Arg; bp 1141: Ser -> Gly). Keep clone #1 for mutation of PstI #1 (design new longer primer).



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