SBB10Ntbk-Paulina Tran
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Paulina Tran 02:00, 9 February 2010 (EST)
Construction file for sbb21
Construction of FokI+ cleavage domain basic part sbb21 PCR fk0001/fk0003 on FokI (505 bp, gp = A) PCR fk0004/fk0002 on FokI (141 bp, gp = B) --------------------------------- PCR fk0001/fk0002 on A+B (620 bp, EcoRI/BamHI) Digest pBjk2741-Bca1144 (EcoRI/BamHI, 2170+910, L) Product is pBjk2741-ssb21 {<FokI+>} --------------------------------- fk0001 Forward EcoRI and BglII for FokI ccaaaGAATTCgtccAGATCTCAGCTGGTTaaatctgaactggaggag fk0004 (F)Base Substitution FokI ccataaaaccaatTgcaatggcgccg fk0003 (R)Base Substitution FokI cggcgccattgcAattggttttatgg fk0002 Reverse BamHI for FokI gctagGGATCCaaaattgatctccccattgttg
Construction file for sbb08
EIPCR tn0001/tn0002R on pBjk2741-Bca1144 (2230 bp, BglII) Product is pBjk2741-ssb08 {Tn5 5’TR} ------- tn0001 EIPCR construction of Tn5 part CCATAagatctGTCGACTGTCTCTTATACACATCTCAACCggatcctaaCTCGCTCCTCaggcttc tn0002R Reverse BglII oligo for Tn5 EIPCR CCAATAGATCTcatgaattcCACTTCAG
17:25, 17 February 2010 (EST)
Today I set up all my 1st step PCRs for sbb21 and ssb08. I labeled the PCR tubes TN0001/0002R, fk 0001/0003 and fk0004/0002.
The PCR procedure is as follows:
1.Dilute Oligos to 100uM (move decimal point for concentration over by one and add that much ddH2O) 2.Make a seperate dilution of 10uM by adding 1uL of the stock to 9uL of water. Mix well. 3.In a PCR tube add 24uL of ddH2O, 3.3uL of 10x expand buffer and dNTPs, 1uL of each of the respective oligos, 0.5uL Template and LASTLY 0.5 uL of the polymerase.
14:30, 22 February 2010 (EST)
Prepped TN0001/0002R for an Analytical gel and FK0001/0003 and FK0004/0002 for the preparative gel.
Ran regular zymo clean up for TN0001/0002R using the following procedure:
1. Add 180uL of Zymo ADB buffer to the 33uL sample of TN 2. Transfered to zymo column 3. Spin in centrifuge at max speed for 30s. Discard the waste. 4. Add 200uL of the wash buffer and spin through. 5. Repeat 6. Spin for 90s to dry 7. Elute with 33uL of elution buffer.