User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/14: Difference between revisions

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Line 38: Line 38:
| bgcolor=#cfcfcf | (x9)
| bgcolor=#cfcfcf | (x9)
| rowspan="7" | Expected:<br>1-8. MV10 = 5191
| rowspan="7" | Expected:<br>1-8. MV10 = 5191
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH041415_gel1.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| Template || 0.2 || 1.8
| Template || 0.2 || 1.8
Line 79: Line 79:
| bgcolor=#cfcfcf | Rxn3,4
| bgcolor=#cfcfcf | Rxn3,4
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH041415_gel2.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| Template || 0.2 || 3.0
| Template || 0.2 || 3.0
Line 107: Line 107:
* Gal4DB-mCh - success!  
* Gal4DB-mCh - success!  
** purify with Sigma PCR purification kit
** purify with Sigma PCR purification kit
** Elute $ back-elute w/ 25 μL elution sln.
** Elute & back-elute w/ 25 μL elution sln.
* ATF2 - failed
* ATF2 - failed
** Try again with 1:10 and undiluted template
** Try again with 1:10 and undiluted template
Line 138: Line 138:
| bgcolor=#cfcfcf | (x9)
| bgcolor=#cfcfcf | (x9)
| rowspan="7" | Expected:<br>1-8. MV10 = 5191
| rowspan="7" | Expected:<br>1-8. MV10 = 5191
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH041415_gel3.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| Template || 0.2 || 1.8
| Template || 0.2 || 1.8
Line 150: Line 150:
| Phusion pol. || 0.5 || 4.5
| Phusion pol. || 0.5 || 4.5
|-
|-
| 5x GC buffer || 5.0 || 45.0
| 5x '''GC buffer''' || 5.0 || 45.0
|-
|-
| DMSO || 1.5 || 7.5
| '''DMSO''' || 1.5 || 7.5
|-
|-
| dH<sub>2</sub>O || 39.8 || 358.2
| dH<sub>2</sub>O || 39.8 || 358.2
Line 166: Line 166:


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




Line 181: Line 182:
| bgcolor=#cfcfcf | Rxn3,4
| bgcolor=#cfcfcf | Rxn3,4
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | No visible product in any lanes
|-
|-
| Template || 0.5 || 2.0
| Template || 0.5 || 2.0
Line 207: Line 208:


Conclusion
Conclusion
* tba
* Failed - order ATF2 cDNA, use as new template for PCR




Revision as of 13:20, 16 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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




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