SBB12Ntbk-John Seggman

John Seggman 02:23, 15 February 2012 (EST)

This is my next entry.

John Seggman 14:33, 16 February 2012 (EST)

Made 100uM stocks of oligos obb1226F and obb1226R

Prepared the following wobble reaction for SBB1226:

29 uL water
5 uL Expand 10x Buffer 2
5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
5 uL Oligo 1 (100uM)
5 uL Oligo 2 (100uM)
0.75 uL Expand Polymerase 1

Made 100uM stocks of oligos araR-F and araR-R For oligos araR-F and araR-R, made an oligo dilution of:

9uL Water
1uL 100uM oligo

Prepared the following PCR reaction for SBB1201:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

Discarded the remainder of the diluted oligos araR-F and araR-R per the "Cloning by PCR" protocol.

Followed all protocols exactly.

John Seggman 16:58, 17 February 2012 (EST)

Performed the Small-Frag Zymo Cleanup on SBB1226:

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

Stored small-frag zymo cleanup product in the freezer for next time.

Next, loaded 6uL of SBB1226 PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel