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 Engineering PC-TFs
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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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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.
- 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.
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Engineering PC-TFs
