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Team:BYU Provo/Team Thermosensor/Week7
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Week 7 (May 29 - June 4)
- June 1
- Sent purified plasmids to sequencing center to check pBAD insert. Also started another PCR for pBAD in case sequences come back negative. Also redoing PCR of LacZ again because of the error we made in restriction sites.
| Tube: | 22-1 | 22-2 | 22-3 | 22-4 |
|---|---|---|---|---|
| rxn: | pBad control | pBAD | LacZ control | LacZ |
- Phusion 50uL Rxn
- ~35 uL ddH2O
- 10uL 5X phusion buffer
- 1.5uL 10mM dNTPs
- 1 uL each primer
- 1 uL diluted template DNA(only in 22-2[PJG408] and 22-4[pIGGY])
- June 2
- PCR cleanup of LacZ and pBAD
- June 3
- Sequenced pLATBAD at the sequencing center and it reconfirmed that our pBAD was not successfully inserted into pLAT, next step: digest pBAD and pLAT for insertion and transformation.
- Performed Restricition digest of pBAD and pLAT at PstI and BamHI on the vector and BglII site on the pBAD insert. (pBAD had a BamHI site in the gene so we couldn’t digest it with BamHI)
- Placed digests in the 370C block in Dr. Grose’s lab
- Dr. Grose would like us to see her for ALL protocol regarding this ligation, it needs to go right.
Lane 1 - DNA Ladder
Lane 2 - RD 23-1
Lane 3 - RD 23-2
lacZ + pBAD PCR
24-1 pBAD
~35 ul ddH20
10 ul 5x Phusion buffer
1.5ul 10 mM dNTPs
1 ul IG 37 (primer)
1 ul IG 38 primer
1 ul PJG 408
0.5 ul Phusion polymerase
24-2 lacZ
~35 ul ddH20
10 ul 5x Phusion buffer
1.5ul 10 mM dNTPs
1 ul IG 40
1 ul IG 41
1 ul PIGGY
0.5 ul Phusion polymerase
24-3 Neg. control
~36 ul ddH20
10 ul 5x Phusion buffer
1.5ul 10 mM dNTPs
1 ul IG 37
1 ul IG 38
Placed in thermocycler
