User:Chia-Hung Tai/Notebook/SBB11/Entry Base

Entry 1: 2/15/11

Prepared for PCR for pBca1766-sbb1141 and pBjh1601KC-sbb1116
-pBca1766-sbb1141
--Phusion is needed since the template is a plasmid DNA
--Instead of adding 3.3 uM 10x of the Expand Buffer, make it 5x by adding 6.6 uM
--Set up the PCR tubes following the protocol

-pBjh1601KC-sbb1116
--Dilute the oligos
--Add 363 uL of water (10 times the concentration) to cht002R
--Add 268 uL of water (10 times the concentration) to cht002F
--Take 1 uL of the mixture for each oligo and add 9 microL of water to get 10 uL
--Set up the PCR tubes following the protocol

Entry 2: 2/17/11

Prepare the PCR products in the analytical gel (since my parts does not require SOEing)
-Start out with 2 0.5 mL tubes
-Add 5 uL of loading dye buffer into each
-Add 2 uL of sbb1141 and sbb1116 PCR product into the respective tubes

Zymo Cleanup (Regular)
-Add 180 uL of Zymo ADB buffer (brown bottle) to a 33 uL reaction.
-Transfer into the Zymo column
-Spin for 30 sec in centrifuge and discard waste. (ALWAYS PUT ON LID!!!)
-Add 200 uL of Zymo Wash buffer
-Spin for 30 sec again and discard waste.
-Add 200 uL of Zymo Wash buffer for second wash
-Spin for 30 sec in centrifuge, discard waste.
-spin for 90 seconds at full speed to dry the column.
-Elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
--Add 33 uL of water to Zymo column
--Spin for 60 sec for water to elute into Eppendorf tube

Stored products in Box B

Entry 3: 2/22/11

According to Chris, we used the wrong wash buffer during the Zymo cleanup (NEED A4 instead of AW). This messed up our product. Chris helped us to redo the PCR products. :D

EcoRI/BamHI Digest of PCR Products
-We need to digest our PCR products

-Prepare 2 PCR tubes, one with sbb1141 product and the other with sbb1116 product
-Add the following to each tubes:
--8 uL of the PCR product
--1 uL of NEB Buffer 2
--0.5 uL EcoRI
--0.5 uL BamHI

-Incubate the tubes at 37 degree C in the thermocycler for 1 hour.

-Run a gel
--Cut out the specific band for your parts (as small as possible)
--Put the gel into the ependorf tube and add 600uL of Zymo ADB buffer
--Heat in the water bath until all the gel has melted
--Put back the products in Box B

Entry 4: 2/24/11

Perform a Zymo Gel Purification
-Transfer into the Zymo column
-Spin for 30 sec in centrifuge and discard waste. (ALWAYS PUT ON LID!!!)
-Add 200 uL of Zymo Wash buffer (MAKE SURE IT IS A4)
-Spin for 30 sec again and discard waste.
-Add 200 uL of Zymo Wash buffer for second wash
-Spin for 30 sec in centrifuge, discard waste.
-spin for 90 seconds at full speed to dry the column.
-Elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
--Add 8 uL of water to Zymo column
--Spin for 60 sec for water to elute into Eppendorf tube

Stored products in Box A

Entry 5: 3/1/11

Performing Ligation on the digests
-Set up the following reaction:
--6.5 uL ddH2O
--1 uL T4 DNA Ligase Buffer
--1 uL Vector digest (for sbb1116: pBjh1601KC; for sbb1141: pBca1766-Bca1089)
--1 uL Insert digest (sbb1116, sbb1141)
--0.5 uL T4 DNA Ligase

-Pound upside down on the bench to mix
-Give it a quick spin to send it back to the bottom of the tube
-Incubate on the benchtop for 30min
-Put on ice and proceed to the transformation

Transforming bacteria through heat shock
-Thaw a 200 uL aliquot of cells on ice
-Add 50 uL of water to the cells
-Add 30 uL of KCM to the cells
-Put your ligation mixture on ice, let cool a minute or two
-Add 70 uL of the cell cocktail to the ligation, stir to mix
-Let sit on ice for 10 min
-Heat shock for 90 seconds at 42 degree C
-Put back on ice for 1 min
-Add 100 uL of LB using sterile technique, let shake in the 37 degree incubator for 1 hour
-Plating --Plate 100 uL sbb1116 on either types of antibiotics plate (both Kan and Cam are fine), Blue-marked plate (Cam) was used
--Plate 100 uL sbb1141 on a special plate of antibiotics (marked yellow, containing spectinomycin??)
--incubate at 37 degrees overnight

Entry 6: 3/3/11

Performed Miniprep for our DNA
Notes: sbb1116 generated 4 separate vials of cells from the dish, but only 1 vial (1 distinct colony detected) for the sbb1141

Procedures for the MiniPrep:
-Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
-Dump supernatant
-If pellet color is pink/red, do not continue further with miniprep for that tube.
-Add 250 uL of P1 (with RNAse) buffer into each tube. Resuspend the cells thoroughly
-Add 250 uL of P2 buffer. Gently mix up and down. Solution should become clearer.
-Add 350 uL of N3 buffer. Tubes become cloudy, so slowly invert a few times, then shake to mix.
-Spin in centrifuge at top speed for 5 minutes.

Using miniprep columns:
Note: Always label every thing that will be used
-Pour liquid from the previous spin into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
-Dump liquid out of the collectors under the columns
-Wash each column with 500 uL of PB buffer.
-Spin in centrifuge at full speed for 15 seconds, then dump out the liquid again.
-Wash with 750uL of PE (make sure ethanol is included) buffer.
-Spin in centrifuge at full speed for 15 seconds and dump out liquid.
-Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
-Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
-Spin in centrifuge at top speed for 30 seconds.
-Take out columns and cap the tubes.

Products stored in Box D

Entry 7: 3/8/11

Mapping the miniprep products

-Prepare 5 PCR tubes with the following content:
--4 uL ddH2O
--4 uL Miniprepped plasmid
--1 uL 10x NEB Buffer 2
--.5 uL EcoRI
--.5 uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
-Since there are 5 minipreps, a master mix was made (amount above x5)
-6 uL total should be added into each PCR tube (only did 5.5 uL though)
-Incubate at 37 on the thermocycler for 30 minutes

-Check for the product from each tube by loading them in gels run the analysis

Entry 8: 3/10/11

Products seem to have the correct base pairs for both sbb1116 (all 4) and sbb1141 (just 1)

The products prepared from last lab were sent for sequencing (2 from sbb1116 and 1 from sbb1141)

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