User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/14

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngKarmella's BioBrick Cloning Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

04/14/15

  • LCR Development



LCR Protocol Development

  • Refer to entry 04/06/2015
  • Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
  1. Gal4DB-mCh (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1 (Tm = 47), R - mCh r1 (Tm = 56)
  2. ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1 (Tm = 56) , R - ATF2_r1 (Tm = 58)
  3. MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1 (Tm = 64), R - MV10 r1 (Tm = 69)


MV10 - Gradient PCR to enhance 5 kb band

  1. 68°C
  2. 67.3°C
  3. 65.9°C
  4. 63.9°C
  5. 61.4°C
  6. 59.6°C
  7. 58.1°C
  8. 57°C
Reagent Rxn1-8 (x9) Expected:
1-8. MV10 = 5191
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.8
10 uM fwd primer 1.0 9.0
10 uM rev primer 1.0 9.0
10 mM dNTPs 1.0 9.0
Phusion pol. 0.5 4.5
5x HF buffer 5.0 45.0
dH2O 41.3 371.7
  50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Failed
  • Try again with 5x GC buffer (more robust amplification) and DMSO (template is 53.7 GC)


Gal4DB-mCh - plasmid
ATF2 - 1:100 iPSC cDNA

  • Run 2 rxns each
Reagent Rxn1,2 Rxn3,4 Expected:
1,2. Gal4DB-mCh = 1170
3,4. ATF2 = 906
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 3.0
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x HF buffer 5.0 5.0
dH2O 41.3 38.5
  50.0 50.0

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 30 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Gal4DB-mCh - success!
    • purify with Sigma PCR purification kit
    • Elute & back-elute w/ 25 μL elution sln.
  • ATF2 - failed
    • Try again with 1:10 and undiluted template
    • Use 5x GC buffer to boost amplification


Measure conc.'s - Gal4DB-mCh PCR

Sample OD260 260/280 ng/μL
1. 5'p-Gal4DB-mCh 0.01 1.23 9.68


MV10 Trial 2 - Gradient PCR to enhance 5 kb band

  1. 68°C
  2. 67.3°C
  3. 65.9°C
  4. 63.9°C
  5. 61.4°C
  6. 59.6°C
  7. 58.1°C
  8. 57°C
Reagent Rxn1-8 (x9) Expected:
1-8. MV10 = 5191
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.8
10 uM fwd primer 1.0 9.0
10 uM rev primer 1.0 9.0
10 mM dNTPs 1.0 9.0
Phusion pol. 0.5 4.5
5x GC buffer 5.0 45.0
DMSO 1.5 7.5
dH2O 39.8 358.2
  50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Failed - try using XbaI-linearized vector as template
    • XbaI cuts in between the two primers, which face outward from the XbaI site


ATF2

  1. 1:1 iPSC cDNA
  2. "
  3. 1:10 iPSC cDNA
  4. "
Reagent Rxn1,2 Rxn3,4 Expected:
1,2. Gal4DB-mCh = 1170
3,4. ATF2 = 906
No visible product in any lanes
Template 0.5 2.0
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x GC buffer 5.0 5.0
dH2O 41.0 39.5
  50.0 50.0

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 30 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Failed - order ATF2 cDNA, use as new template for PCR