User:Alexander Boehme/Notebook/BioE140L

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Take out columns and cap the tubes.
Take out columns and cap the tubes.
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.</pre>
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.</pre>
 +
 +
Then do restriction map w/ EcoRI & XhoI
 +
Set up the following 10uL reaction in a PCR tube:
 +
6 uL ddH2O
 +
2uL Miniprepped plasmid
 +
1uL 10x NEB Buffer 2
 +
0.5uL EcoRI
 +
0.5uL XhoI
 +
Incubate at 37 on the thermocycler for 30 minutes
 +
Run an analytical gel
 +
Take a picture of the gel
 +
Calculate the expected fragment sizes
 +
Are the calculated sizes consistent with the bands on the gel?

Revision as of 13:01, 11 April 2013

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3/6/2013: Designed construction file for Overall Project

AsbF-2 Synthon Construction File


Mix oligios for asbF-2 synthon, do PCA1 protocol              (514 bp, pca1)
 
PCR pca1 with asbF2-NewForward and asbF2-NewReverse by PCA2 protocol         (468 bp, pca2)
 
Digest pca2 with EcoRI/BglII (yes, BglII), gp                 (452+11+5 bp, pcr_dig)
 
(get pre-digested vector from JCA of pBca9145-Bca1144  (EcoRI/BamHI, vect_dig)
 
Ligate pcr_dig and vect_dig, transform, pick white colonies, map, sequence

asbF2-NewForward
ccaaaGAATTCCGTCTCATAGAAACCCATCC

asbF2-NewReverse
gactgAGATCTCGTCTCAGGTCTCAGGATCCAG 


"'3/9/2013: PCA1 Performed

PCA1 Protocal
38 uL ddH2O
5 ul 10x expand buffer
5 ul 2mM dNTPs
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
0.75 ul Expand polymerase
Program (can run JCA/PCA1)
2 min initial denature at 94oC
30 sec denature at 94oC
30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
30 sec extension at 72oC
repeat 2-4 30 times total

"'3/12/2013 Regular Zymo Cleanup on PCA1

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 Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo 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 as the volume of the original reaction

Then perform PCA2:

''Amplification'''
Now, you need to do an amplification of the correct full-length chunks. Clean up the assembly reaction with a zymo column; don't bother running it on a gel - it'll be a smeary mess and won't really help you. Save the purified product in case this step fails! For the amplification reaction, do a normal phusion program with 1 ul of the cleaned up assembly reaction as template, and using the outermost oligos for the chunk. That is:
'''Recipe'''
1 ul each outer oligo (10 uM)
   -Dilute F/R oligos 1:10 from 100uM; in this case, oligo CCOMT1-12/CCOMT1-15
1 ul purified pca product
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O

Samples given to John to run the Program. 1:10 Oligo12/Oligo15 dilutions in small PCR tubes. Also stored purified PCA1 product in 4o box.

'''Program'''
2 min initial denature at 94oC
30 sec denature at 94oC
30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
30 sec extension at 68oC
repeat 2-4 30 times total

"'3/14/2013 Gel from all class samples. Gel is of PCA2 products for various synthons. Lane1=His-tag part's IPCR product, Lane2=Molecular weight standards Rest of the lanes are PCA2's


Lane 5 is ASFB-2 (mine) 2013_03_13-JCA-gel1.jpg


Zymo CleanUp on PCA2 product
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 Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo 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 as the volume of the original reaction

EcoRI/BamHI Digest on PCA2
1. 1uL of NEB Buffer 2 (ADD THIS FIRST)
2. 8uL of eluted PCR product
3. 0.5uL EcoRI
4. 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
Zymo Gel Purification
1. Add 2uL Loading Dye to 10uL sample
2. Load all of sample into well
  -Gel "R" lane 5
3. Run gel @ 180V (Maximum before temperature heats gel to melt) ~20 minutes
4. Cut band out and incubate in 300uL ADB buffer until gel melted
5. Place in Fridge

"'3/19/2013

Zymo Cleanup after Gel Purification
1. transfer into the Zymo column inside a collection tube (small clear guys)
2. spin through, discard waste.
3. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
4. spin through, discard waste.
5. Add 200 uL of Zymo Wash Buffer
6. spin through, discard waste.
7. spin for 90 seconds, full speed to dry.
8. elute with water into a fresh Eppendorf tube. Water amount should be same as DNA input - '''8uL'''

Ligation -> Heat Shock -> Plating

Ligation of EcoRI/BamHI digests

 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

Transformation Take competent cells to which TSS has been added. Heat shock Rescue with 2YT

Thaw a 200 uL aliquot of cells on ice
Add 50 uL of water to the cells (if greater volume is desired)
Add 30 uL of KCM to the cells 
Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
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 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DID NOT RESCUE FOR 1 HOUR BECAUSE AMPICILLIN)
Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight



'4/2/2013

Issue with colonies, could be issue with restrictive enzymes, buffers used, ran only a small amount of gel. 
Therefore redid PCA2 pdt cleanup
Protocol
EcoRI/BglII Digest on PCA2
1. 1uL of NEB Buffer 2 (ADD THIS FIRST)
2. 8uL of eluted PCR product
3. 0.5uL EcoRI
4. 0.5uL BgIII

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

In the meantime run gel purification on PCR pdt digestion
Zymo Gel Purification
1. Add 0.5 uL Loading Dye to 2uL sample
2. Load all of sample into well
Loaded to lane 4 (2nd gel)

Then did regular zymo clean up on the PCA2 that was digested with EcoRI/BglII (added 8µL of water to eppendorf tube to elute)

4/4/2013 Redid what we did on 3/19 so Ligation -> Heat Shock -> Plating

Ligation of EcoRI/BamHI digests

 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

Transformation Take competent cells to which TSS has been added. Heat shock Rescue with 2YT

Thaw a 200 uL aliquot of cells on ice
Add 50 uL of water to the cells (if greater volume is desired)
Add 30 uL of KCM to the cells 
Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
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 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DID NOT RESCUE FOR 1 HOUR BECAUSE AMPICILLIN)
Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

"'4/9/13 Colonies did not quite work for me, but I'm going to try using one of the white colonies (there was only one white colony). Did not do colony PCR, so only added 5mL of media to culture tubes (most people had 4, I only had 1). Picked out white colony with pipette tip then placed tip into culture tube.

4/11/2013

Miniprep purification of DNA
MINIPREP (2mL) Procedure for Plasmid DNA Purification
(using the QIAGEN QIAPrep Spin Miniprep kit)
!!!!! Make sure Ethanol has been added to the PE Buffer !!!!!
!!!!! Make sure that RNAse has been added to the P1 Buffer !!!!!
Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. (Did this by first doing 1mL, spin, get rid of supernatant, add 1mL again, then spin again)
Dump supernatant
Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
Spin in centrifuge at top speed for 5 minutes.
Label blue columns with an alcohol-resistant lab pen.
Pour liquid 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 (the DNA should be stuck to the white resin)
Wash each column with 500 uL of PB buffer.
Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
Wash with 750uL of PE buffer (washes the salts off the resins).
Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
Label new Microcentrifuge tubes and put columns in them.
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.
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Then do restriction map w/ EcoRI & XhoI Set up the following 10uL reaction in a PCR tube: 6 uL ddH2O 2uL Miniprepped plasmid 1uL 10x NEB Buffer 2 0.5uL EcoRI 0.5uL XhoI Incubate at 37 on the thermocycler for 30 minutes Run an analytical gel Take a picture of the gel Calculate the expected fragment sizes Are the calculated sizes consistent with the bands on the gel?

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