Haynes Lab:Notebook/Engineering PC-TFs/2012/12/06

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Summary

Dilute primers that came from IDT (10x dilution of concentration)

  • BL_Primer09 26.6 nm --> 266 μL dilution
  • BL_Primer10 25.7 nm --> 257 μL dilution
  • Thaw and dilute primers that came (x10 dilution)
  • 90 μL water
  • 10 μL primer stock

(10 μm in each tube for PCR reaction)
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  • Assemblies

1. hPCD - Pflex - BL01
2. hPCD - BL01

# Template Primers
1 hPCD 9, 2
2 Plflex 3, 4
3 BL01 5,10
4 hPCD 9, 7
5 BL01 8,10
μL
Template .5 μL
Primer 1 (10 μm) 1.0 μL
Primer 2 (10 μm) 1.0 μL
2*GoTaq 12. 5
DH2O 10.0 μL

Run PCR with preset GoTaq settings. Need to ask Dr. Haynes to be certain settings are correct for my reactions.
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*Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~10 min.
  • Vi helped me with running gel because I had class. Vi will put digest into gel and will get results.
  • I believe that I did not wait long enough after I microwaved the gel to put in the ethnium bromide. I need to be sure that I wait until its cooled down.



PSB1A3 VECTOR digest
  • Vi cut gel and has it available for me to work on tomorrow afternoon.
  • Vi also took out my PCR reaction. She told me that the lid was not closed all the way, so I may need to redo rxns.