Arking:JCAProtocols

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===LR Gateway Transfers===
 
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Revision as of 17:03, 22 January 2014

J. Christopher Anderson, Assistant Professor, UC Berkeley



Assembly Reactions

Golden Gate Assembly

Reaction mix:

2uL of each plasmid*
1uL T4 DNA ligase buffer
0.5uL T4 DNA ligase
0.5uL BsaI
6uL water
10uL total volume

Ideal is equimolar amounts of each plasmid, but if your mini preps are consistent, you don’t really have to bother normalizing.

Thermocycler program:

37C for 2min
16C for 5min
Repeat 25 times*
45C for 10min
80C for 10min
10C hold

Our default is 25 cycles. You can reduce the number of cycles (we often do 15) to shorten the reaction time, but efficiency goes down. We’ve never tried doing more than 25 cycles

LR Gateway Transfers

In preparation for doing the Gateway reaction, check out the following page:
http://2008.igem.org/Team:UC_Berkeley/GatewayOverview

You will be doing a normal in vitro LR gateway reaction. The pBca1254** vectors all contain the attR1 and attR2 which will recombine with the attL1 and attL2 sites in your donor pBca1256 plasmids. Upon reaction, the lethal ccdB gene in the pBca1254** assembly vector will be displaced allowing for selection of your product after transformation. The name of your product plasmid will be pBca9495**-partname. There are no restriction enzymes, ligases, gels, or zymo columns in the Gateway reaction! You just mix plasmids and enzyme, let it cook, then transform

  • Set up the following mixture in a 0.5mL microcentrifuge tube
 3uL ddH2O
 0.5uL Donor plasmid (a pBca1256-*)
 0.5uL Recipient plasmid (a pBca1254**-*)
  • Add 1uL of LR Clonase
  • Slam on bench upside down to mix
  • Quick spin to send it to the bottom of the tube
  • Incubate at 25 degrees on the thermocycler for 1hr
  • Add 0.5uL proteinase K
  • Slam on bench upside down to mix
  • Quick spin to send it to the bottom of the tube
  • Incubate at 37 degrees on the thermocycler for 10min.
  • Put on ice, proceed to transformation

PCR Reactions

Wobble Reaction

The Wobble procedure is a variation of Klenow Extension that begins with two oligonucleotides that overlap by around 20 bp on their 3' ends and uses a thermostable polymerase

  • Order the oligos, they don't need to be purified in any special way, smallest scale is ok
  • Make 100uM stocks, this is the concentration used directly for the reaction
  • Prepare the following reaction:
 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
  • Run the wobble program, whick is:
 2 min at 94
 10 cycles of:
 30 sec at 55
 30 sec at 72
 (or something similar)
  • There is no point in running an analytical gel afterwards, there is nothing to see
  • You'll want to run short fragment cleanups to remove the polymerase prior to digestion steps

Cloning by PCR

This is the basic PCR method to amplify your DNA from a plasmid using the Phusion polymerase. You also use this protocol for an EIPCR reaction.

The oligo concentrations in your stocks should be 100uM. You use them at 10uM in this protocol. So, you first need to make an oligo dilution of:

 9uL Water
 1uL 100uM oligo

You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube!

Set up the following reaction in a PCR tube:

31.5uL ddH2O
10uL 5x Phusion Buffer
5uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
1uL Template DNA
0.5uL Phusion Polymerase

The Expand temperature programs are a little complicated, so I won’t write them out here. They all involve 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.

SOEing PCR

  • Set up PCR reactions according to your construction file as normal 33uL reactions as described in Cloning by PCR
  • For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel
  • Cut out the bands, put them into a single 1.5mL microcentrifuge tube
  • Add 650uL of ADB Buffer
  • Proceed with the Zymo Gel Purification procedure
  • Elute the DNA in 50uL of water
  • Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template

CleanupReactions

Regular Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.


  1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, discard waste.
  4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Small-Frag Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

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

Zymo Gel Purification

  • 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 Zymo Wash Buffer (which is basically 70% ethanol)
  8. spin through, discard waste.
  9. Add 200 uL of Zymo 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

DigestionsReactions

EcoRI/BamHI Digest of Wobble Products

For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

  • Set up the following reaction in a PCR tube:
 50uL eluted DNA
 5.7uL NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI
  • Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
  • Incubate the reaction at 37 degrees on the thermocycler
  • Proceed to another Zymo small fragment cleanup

EcoRI/BamHI Digest of PCR Products

For PCR products, you will only digest a portion of your purified PCR product.

  • 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

BglII Digest of EIPCR Products

For EIPCR products, you will only digest a portion of your purified PCR product. This procedure is almost identical to the digestion of a PCR product with EcoRI/BamHI, but you'll use BglII instead of EcoRI/BamHI.

  • Set up the following reaction:
 8.5uL of eluted PCR product
 1uL of NEB Buffer 2
 1uL BglII
  • Incubate at 37 degrees on the thermocycler for 1hr
  • Run an agarose gel, cut out the band, and melt with 600uL ADB buffer at 55 degrees
  • Proceed with remainder of Zymo gel purification protocol

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

Ligation of EIPCR digests

  • Set up the following reaction:
 7.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL PCR product 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

BacteriaManipulations

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

  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

Picking of colonies

  • For each construct you will pick and later miniprep 2 colonies
  • Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  • Pick a well-isolated, round, and "normal" looking colony with a toothpick
  • Drop it in the test tube
  • Incubate at 37 overnight

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 !!!!!

  1. Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
  2. Dump supernatant
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. 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.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen.
  8. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
  9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  10. Wash each column with 500 uL of PB buffer.
  11. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
  12. Wash with 750uL of PE buffer (washes the salts off the resins).
  13. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
  14. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  15. Label new Microcentrifuge tubes and put columns in them.
  16. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
  17. Spin in centrifuge at top speed for 30 seconds.
  18. Take out columns and cap the tubes.
  19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.
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