SBB11Ntbk-JosephSilo

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Joseph Silo 8 March 2011 (EST)

Analytical mapping of the mini-prepped products was performed.

Followed this protocol:

-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


Image:030811-Megagel.png 1 through 8 may be inverted, though.

Joseph Silo 18:06, 3 March 2011 (EST)

Colonies were already picked and innoculated when I arrived to lab. There were four replicates of each part. The Miniprep protocol was used Template:SBB-Protocols_Micro3.

Joseph Silo 18:03, 1 March 2011 (EST)

Ligation and transformation of our PCR products were done.

The ligation protocol is:

Ligation of EcoRI/BamHI digests 
Set up the following reaction:
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL 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

The transformation protocol is:

1. Thaw a 200 uL aliquot of cells on ice 
2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

Joseph Silo 13:00, 24 February 2011 (EST)

Completed the Zymo Gel Purification step on sbb1120, sbb1131, and sbb1105. Eluted each with 8 microliters of water.

The protocol is:

All spins until the drying step are 15 second full speed spins. 
1.cut out bands minimizing extra gel matter.
2.put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3.heat at 55, shake and/or vortex until the gel has dissolved.
4.If the DNA is <300bp add 250uL of isopropanol
5.transfer into the Zymo column inside a collection tube (small clear guys)
6.spin through, discard waste.
7.Add 200 uL of A4 Wash Bbuffer (which is basically 70% ethanol)
8.spin through, discard waste.
9.Add 200 uL of A4 Wash Buffer
10.spin through, discard waste.
11.spin for 90 seconds, full speed to dry.
12.elute with water into a fresh Eppendorf tube

Joseph Silo 12:55, 21 February 2011 (EST)

Dr. Anderson redid all of the PCR products because people may have used the wrong buffer during the Regular Zymo Cleanup step. Did an EcoRI/BamHI Digest of PCR Products sbb1120, sbb1131, and sbb1105. The protocol for this procedure is:

Set up the following reaction: 
8uL of eluted PCR product
1uL of NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI
Incubate at 37 degrees on the thermocycler for 1hr
Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column


Ran an agarose gel (Originally placed in "Gel C" with sbb1120, sbb1131, and sbb1105 in C7, C8, and C6, respectively. This gel may have been moved and/or renamed as the following gel pics below).


Image:022111-AnalGel3.jpg

sbb1120 is in Lane 13. Expected basepairs: 1031



Image:022111-AnalGel4.jpg

sbb1131 is in Lane 2. Expected basepairs: 1084



Image:022111-AnalGel3.jpg

sbb1105 is in Lane 7. Expected basepairs: 986

Joseph Silo 14:25, 17 February 2011 (EST)

Did an analytical gel for PCR parts sbb1120, sbb1131, and sbb1105. PCR parts are in Analytical Gel Pic 3, in lanes 8, 9, 10, respectively. See image below.

Image:021711-AnalGel3.jpg

Bands are good.

Did a Regular Zymo Cleanup:

Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
Spin through, discard waste.
Add 200 uL of A4 Wash Buffer (which is basically 70% ethanol)
Spin through, discard waste.
Add 200 uL of A4 Wash Buffer
Spin through, discard waste.
Spin for 90 seconds, full speed to dry.
Elute with water into a fresh Eppendorf tube, use the same volume of water (8μL) as the volume of the original reaction
That is, I eluted with 33 microliters of water.

Joseph Silo 14:10, 15 February 2011 (EST)

PCR reactions were set up for parts sbb1120, sbb1131, and sbb1105 using MG1655 genomic DNA as templates. The [[1]] "Cloning by PCR" protocol was used.

Joseph Silo 23:49, 14 February 2011 (EST)

I'm ready.

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