Electroporation Protocol

2015-01-13

azim58 - Electroporation Protocol

Electroporation Protocol 081711
adapted from Molecular Cloning Book Electroporation Protocol (Protocol
26: Transformation of E. coli by Electroporation 1.121)

* Materials
  o Cold electroporation cuvettes
  o Cold 1.5 mL tubes
  o DNA (include positive (like PUC19) and negative controls (H20))
  o Competent cells
  o Pipettes (2 uL, 20 uL)
  o Pipette tips for 2 and 20 uL pipette
  o 200 uL pipette for long tips
    - last seen on Valiery's old bench

o Long tips (1-200 uL Gel loading tips Cat No 1022-0000)
    - last seen above my bench

o Pipette tip waste container
  o Kim wipes
  o SOC
  o 1.5 mL tube for incubation
  o tube holder for 1.5 mL tube
  o H20 (only if a negative control is included)
  o LB antibiotic plates dried before use (may be best to dry the
    night before use)

* -Start drying plates in 37 C incubator the night before or a few
  hourse before
* -Transfer cells to ice-cold sterile 1.5 mL microfuge tubes.
  o -20 uL cells per sample is sufficient

* -Set the electroporation apparatus to deliver an electrical pulse
  appropriate for the cuvette size used.
  o -0.2 cm cuvette: 2.5 kV, 25 uF, 200 Ohm
  o -0.1 cm cuvette: 1.8 kV, 25 uF, 200 Ohm

* -Add DNA in a volume of 0.5-2.5 uL (smaller volumes are better)
  (dilute in water so that as little salt is used as possible). May want
  to heat the sample at 60 C before use since this seems to improve
  transformation efficiency.
  o 10 pg (for PUC19 positive control) to 100 ng of the DNA should be
    added.
    Source:http://tools.invitrogen.com/content/sfs/manuals/18290015.pdf.
    Note that if DNA from a ligation or In-Fusion is used the
    transformation efficiency calculated later on should correspond to
    the amount of vector used rather than the total amount of DNA in the
    solution.
  o some protocols recommend that the DNA should be diluted in TE
    buffer
  o As of 4-25-13 I'm thinking I may want to heat DNA at 50-60 C for
    a little while to loosen all the DNA up from the glycogen. Then I
    could cool the DNA down a little, and then do the electroporation.

* -Pipette the DNA/cell mixture into a cold electroporation cuvette.
  Tap the solution to ensure that the suspension of bacteria and DNA sits
  at the bottom of the cuvette. Dry condensation and moisture from the
  outside of the cuvette. Place the cuvette in the electroporation
  device. Avoid the formation of bubbles.
  o -PUC19 plasmid: 10 pg
  o -Other Plasmid: usually 1-10 ng

* -Deliver a pulse of electricity to the cells at the settings
  indicated above. A time constant of 4-5 milliseconds should register on
  the machine.
* -As quickly as possible after the pulse, remove the electroporation
  cuvette and add 3-5X the more room temperature SOC than there are
  cells. For example, for 20 uL cells add 100 uL SOC
* -Transfer the cells to a 1.5 mL tube and incubate the cultures at 250
  rpm 37 C 1 hr (or 40 min to prevent any additional unwanted cell
  divisions).
* -Pipette culture onto plate and spread with cell spreader.-Let the
  culture soak into the plate at room temperature for 10-20 min
  o -Use 50-200 uL cutlure (alternatively plate 10 uL onto one plate
    and 90 uL onto another)
  o -May want to prepare multiple dilutions of the cells to plate
    e.g. 1:100, 1:10,000, etc.

* -Invert the plates and incubate them at 37 C. Transformed colonies
  should appear in 12-16 hours.

---------
Cleaning electroporation cuvettes
-I generally try not to reuse electroporation cuvettes because the
probability of arcing is much higher. However, I clean the cuvettes for
potential reuse by washing with water, then 70% ethanol, then drying, and
then exposing to UV.

-Electroporation FAQ here
http://www.btxonline.com/pages/FAQ.html#l
-Exponential decay waves will result in a higher rate of cell mortality
in mammalian cells and square waves will result in lower transformation
efficiencies in bacteria and yeast.

-Can DNA in TE or just Tris buffer be used with electroporation?
--Yes it can, but transformation efficiency is reduced compared to water.
I tried 10 mM Tris previously. see:
"F:\kurt\storage\CIM Research Folder\DR\2013\5-10-13\Transformation
Results 5-10-13.xlsx"
--Yes it can. Generally you want the lowest concentration of salt
possible though. Here's a link where they mention this
science.marshall.edu/murraye/340/Analyzing%20Data.ppt
TE buffer is even recommended in some protocols
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006174B.pdf
http://userpages.umbc.edu/~jwolf/m7.htm
http://www3.imperial.ac.uk/lifesciences/services/research/transgeniclist/pr
otocols/electropprotocol
--Some protocols seem to recommend against it.
---http://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=we
b&amp;cd=8&amp;ved=0CG4QFjAH&amp;url=http%3A%2F%2Feshop.eppendorfna.com%2Fd
ocuments%2Fdownload%2FBrochure_Multiporator_Multiporator_Succ_163997_Brochu
re&amp;ei=l2t5UbaRJO75igLfv4DoBw&amp;usg=AFQjCNGoJjQEEStjy_4qASoaJEe_eFu6QA
&amp;sig2=MCNJsuW5FKET2fCUEni6DA&amp;bvm=bv.45645796,d.cGE&amp;cad=rja
----Contamination caused by endotoxins, EDTA (e.g. in TE buffer) or high
salt concentrations can decrease the transfection efficiency.
----It should be ensured that the low osmolarity of the electroporation

-Heating DNA solution
http://bitesizebio.com/articles/dna-precipitation-ethanol-vs-isopropanol/
For dry DNA pellets, heating the sample in buffer at 50-60C will help the
DNA dissolve faster and won’t damage the DNA. For RNA, heating can be
used too (in water) at temps around 42C. Overdried DNA and RNA will take
longer to dissolve so make sure not to speed vac for too long.
--In one test heating generally seems to improve my transformation
efficiency. see:
"F:\kurt\storage\CIM Research Folder\DR\2013\5-10-13\Transformation
Results 5-10-13.xlsx"
There was a (5.47E9 cfu/ug)/(4.44E8 cfu/ug)=12.3 fold difference from the
G+H-T- to the G+H+T- sample. If this result is repeatable, this indicates
that using heat with glycogen and H20 significantly increases
transformation efficiency compared to not using heat.
--Kathy thinks she may have heated at about 72 C, but this value was just
off the top of her head.

-Does glycogen in a solution decrease transformation efficiency?
Glycogen seems to decrease transformation efficiency slightly, but not
too much. see:
"F:\kurt\storage\CIM Research Folder\DR\2013\5-10-13\Transformation
Results 5-10-13.xlsx"

Does it make a difference to pipette the cells with DNA, leave the DNA
with the cells for 1 min, or use the cells immediately (1-10 s) after
adding DNA?
see DNA incubation time 11-14-12
basically the answer to this question is "not really"

Do ethanol precipitated in-fusion products transform better with chemical
transformation or electroporation?
Electroporation seems to work better, but there are some caveats
see
"C:\kurt\storage\CIM Research
Folder\DR\2013\2-6-13\transformation_efficiency\Transformation Efficiency
Test 2-6-13.xlsx"

Does diluting the glycogen precipitated in-fusion product make a
difference?
Not really
"C:\kurt\storage\CIM Research
Folder\DR\2013\2-6-13\transformation_efficiency\Transformation Efficiency
Test 2-6-13.xlsx"

Does it make a difference to incubate the cells in 1 mL SOC as opposed to
about 0.1 mL SOC?
Not really
"C:\kurt\storage\CIM Research
Folder\DR\2013\2-6-13\transformation_efficiency\Transformation Efficiency
Test 2-6-13.xlsx"

How old can carb plates be? 11-7-12

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