SBB11Ntbk-NikitPatel

=Constructing Basic Parts=

Nikit Patel 11:30, 15 February 2011 (EST)
Thought of the day: Did my first PCR after 3 years at Berkeley!


 * Create 100uM stock of oligos: P_sbp_inR and SS50r
 * Followed Cloning by PCR Protocol:
 * - Used construction files below to setup PCR
 * - Did first part of SOEing for P_sbp basic part
 * - Thermocycler Program: 2K55

Construction of P_sbp BglBrick basic part sbb1124 PCR ss50f/P_sbp_inR on MG1655	(247 bp, gp = A) PCR P_sbp_inF/ss50r on MG1655	(492 bp, gp = B) --- PCR ss50f/ss50r on A+B	               (710 bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144 		(EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1124          {P_sbp} ---			P_sbp_inR	Reverse removal of EcoRI site in P_sbp CAATAAATTGCAGAAGTCATGTAGGCCTG P_sbp_inF	Forward removal of EcoRI site in P_sbp CAGGCCTACATGACTTCTGCAATTTATTG ss50f	sbp aaaccGAATTCatgAGATCTgcggtcgttgtgtaggtatccag ss50r	sbp tttggGGATCCcaacatcagcttcaataccgttg

Part sbb1104                           {P_fadL} PCR ss29f/ss29r on MG1655 gen.         (611bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144#5          (EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1104           {P_fadL} --- ss29fForward Cloning of P_fadL    aaaccGAATTCatgAGATCTgctttttcagtcagcgccgccag ss29rReverse Cloning of P_fadL    tttggGGATCCgataagtgccactgcgactgcgagagc

Nikit Patel 12:35, 17 February 2011 (EST)
''Thought of the day: Is it me or is ADB buffer like magic? It annihilated the gel so fast!''


 * P_sbp Products A and B
 * - Ran preparative gel
 * - Lanes 2 and 3 from gel (below) confirmed sizes around 250 and 500 bp respectively
 * - Bands were cut
 * - Performed Zymo Gel Purification on both A + B
 * - Setup and run PCR for SOEing using Zymo Gel Purified DNA as template. (Used 2K55 mode for thermocycler)


 * [[Image:021711-PrepGel1.jpg | 250 px]]


 * P_fadL Products
 * - Ran analytical gel (prepped 2uL DNA + 5uL Dye)
 * - Band in lane 2 from gel (below) confirmed size around 600 bp
 * - Perform Regular Zymo Cleanup
 * [[Image:021711-AnalGel2.jpg | 250px]]

Nikit Patel 18:17, 18 February 2011 (EST)
Thought of the day: Nikit + Gary = Best SOEing partners!


 * P_sbp SOEing PCR products
 * - Ran analytical gel
 * - Lane 2 from gel (below) confirmed size around 750 bp. (SOEing worked!)
 * - Performed Regular Zymo Cleanup
 * [[Image:021811-AnalGel1.jpg | 250 px]]

Nikit Patel 14:29, 22 February 2011 (EST)
''Thought of the day: Shout out to Professor Anderson for redoing our PCRs. We will never forget this.''


 * PCRs were redone. Analytical Gel Lane 14 (below) confirms successful PCR product
 * [[Image:022111-AnalGel2.jpg | 250 px]]


 * P_sbp (Part sbb1124)
 * - Performed analytical gel
 * - Lane 1 below confirms SOEing worked
 * [[Image:022211-AnalGel1.jpg | 250 px]]


 * P_fadL (Part sbb1104)
 * - Performed EcoRI/BamHI Digest
 * - Digest run for 1 hour in thermocycler
 * - Run preparative gel (below)
 * - Added 600 uL of ADB buffer to Lane 5 band
 * [[Image:022211-PrepGel5.jpg | 250 px]]

Nikit Patel 13:48, 24 February 2011 (EST)
''Thought of the day: It's Vini's birthday today! I also found out that Gary is a funny guy.''


 * P_sbp (Part sbb1124)
 * - Gels were cut and loaded with ADB buffer by Professor Anderson the day before
 * - Performed EcoRI/BamHI Digest
 * - Digest run for 1 hour in thermocycler
 * - Ran preparative gel. Lane 1 is my band! (below)
 * - Performed rest of Zymo Gel Purification
 * - Labelled as "sbb1124 Cut and Cleaned"
 * [[Image:022411-PrepGel1.jpg | 250 px]]


 * P_fadL (Part sbb1104)
 * - Performed rest of Zymo Gel Purification
 * - Eluted in 8 uL of water
 * - Labelled as "sbb1104 Cut and Cleaned"

Nikit Patel 14:59, 1 March 2011 (EST)
Thought of the day: Sad that UCB Ph.D candidates are visiting today and I'm not one of them.


 * Performed ligation reaction on both parts
 * - Used pBjh1601KC-Bca1144 vectors for both
 * - Incubated on bench top for 30 minutes


 * Performed transformation by heat-shock on ligation products
 * - Shared cells with Gary
 * - Heat shocked for 120 seconds

Nikit Patel 3:44, 3 March 2011 (EST)
''Thought of the day: Marcus was acting really hyper during lab. It was entertaining!"


 * Plate had many colonies.
 * Four colonies were picked from each plate for us
 * Performed Miniprep Purification on the 8 cultures

Nikit Patel 13:37, 8 March 2011 (EST)
Thought of the day: I want Sergey to add me on Words with Friends!


 * Performed Analytical Mapping Digests on all 8 minipreps
 * Ran the digested products on a gel (below)
 * - Lane #5 : sbb1104 #1
 * - Lane #6 : sbb1104 #2
 * - Lane #7 : sbb1104 #3
 * - Lane #8 : sbb1104 #4
 * - Lane #9 : sbb1124 #1
 * - Lane #10 : sbb1124 #2
 * - Lane #11 : sbb1124 #3
 * - Lane #12 : sbb1124 #4


 * [[Image:030811-Gel2.jpg | 250 px]]

Nikit Patel 13:00, 10 March 2011 (EST)
Thought of the day: BioE 100 midterm is stealing my mind away from BioE 140L midterm!


 * Lane 11 doesn't look good.
 * Therefore, we will be putting in sbb1104 #1/2 and sbb1124 #1/2 for sequencing