SBB10Ntbk-Paulina Tran

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.