SBB11Ntbk-JosephSilo

Joseph Silo 18:06, 6 March 2011 (EST)

March 3. Colonies were already picked and innoculated when I arrived to lab. There were four replicates of each part. The Miniprep protocol was used.

Joseph Silo 18:03, 6 March 2011 (EST)

Ligation and transformation of our PCR products were done on March 1.

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).

sbb1120 is in Lane 13. Expected basepairs: 1031

sbb1131 is in Lane 2. Expected basepairs: 1084

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.

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.