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.

14:30, 22 February 2010 (EST)

Prepped TN0001/0002R for an Analytical gel and FK0001/0003 and FK0004/0002 for the preparative gel.

Lane 10 is TN0001/0002R

Lane 6 is FK 0001/0003 and Lane 7 is FK0002/0004

Analytical gel for TN looked good, so proceeded to zymo clean up.

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.

-Stored TN elution in Box A

The preparative gel for Fk 0001/0003 showed the correct size and was cut from the gel, suspended in 600uL ADB buffer, and melted.

Fk0002/0004 was shown to be a larger size than expected. I repeated the PCR with and with out DMSO. Hopefully I will get the desired product.

14:12, 24 February 2010 (EST)

First I prepped the FK0002/0004 with and without DMSO samples for the preparative gel. I then proceeded to prep my TN0001/0002R for the digestion by adding 8.5uL of eluted PCR product to 1uL of NEB Buffer 2 and lastly adding the enzyme 0.5uL BglII. I incubated the samples at 37 degrees for an hour on the thermocycler.

After incubation, I Prepped the BglII digested TN1/2R for a preparative gel. The band looked good and the DNA was cut out of the gel and melted in ADB buffer.

14:30, 26 February 2010 (EST)

FK 0001/0003 and FK 0002/0004 lost. Remade the FK0001/0003 and FK0002/0004 oligos. Ran preparative gels for each, cut out the segments and melted them in ADB buffer.

14:30, 1 March 2010 (EST)

Prepped TN1/2R for ligation and transformation.

14:30, 3 March 2010 (EST)

Miniprep TN1/2R. Prepped FK 1/3 2/4 for analytical gel.

sbb21 (FK 1/3 2/4) product is expected to have a band at ~600bp. The product is in lane 4 and looks approximately at 600bp. Product is good. Will continue with EcoI/BamHI digest next week.