SBB10Ntbk-Paulina Tran: Difference between revisions
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The PCR procedure is as follows: | The PCR procedure is as follows: | ||
1.Dilute Oligos to 100uM (move decimal point for concentration over by one and add that much ddH2O) | 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. | |||
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. | ||
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. | |||
Revision as of 16:10, 17 February 2010
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.
