Electroporation+Protocol

__Electroporation Protocol 081711__ adapted from Molecular Cloning Book Electroporation Protocol (Protocol 26: Transformation of E. coli by Electroporation 1.121)
 * __Materials__
 * Cold electroporation cuvettes
 * Cold 1.5 mL tubes
 * DNA (include positive (like PUC19) and negative controls (H20))
 * Competent cells
 * Pipettes (2 uL, 20 uL)
 * Pipette tips for 2 and 20 uL pipette
 * 200 uL pipette for long tips
 * last seen on Valiery's old bench
 * Long tips (1-200 uL Gel loading tips Cat No 1022-0000)
 * last seen above my bench
 * Pipette tip waste container
 * Kim wipes
 * SOC
 * 1.5 mL tube for incubation
 * tube holder for 1.5 mL tube
 * H20 (only if a negative control is included)
 * 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.
 * -20 uL cells per sample is sufficient
 * -Set the electroporation apparatus to deliver an electrical pulse appropriate for the cuvette size used.
 * -0.2 cm cuvette: 2.5 kV, 25 uF, 200 Ohm
 * -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.
 * 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.
 * some protocols recommend that the DNA should be diluted in TE buffer
 * 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.
 * -PUC19 plasmid: 10 pg
 * -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
 * -Use 50-200 uL cutlure (alternatively plate 10 uL onto one plate and 90 uL onto another)
 * -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 <span style="font-family: 'Courier New',Courier,monospace; font-size: 90%; vertical-align: middle;">-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/protocols/electropprotocol --Some protocols seem to recommend against it. ---http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&ved=0CG4QFjAH&url=http%3A%2F%2Feshop.eppendorfna.com%2Fdocuments%2Fdownload%2FBrochure_Multiporator_Multiporator_Succ_163997_Brochure&ei=l2t5UbaRJO75igLfv4DoBw&usg=AFQjCNGoJjQEEStjy_4qASoaJEe_eFu6QA&sig2=MCNJsuW5FKET2fCUEni6DA&bvm=bv.45645796,d.cGE&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"

<span style="font-family: 'Courier New',Courier,monospace; font-size: 90%; vertical-align: middle;">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"

<span style="font-family: 'Courier New',Courier,monospace; font-size: 90%; vertical-align: middle;">How old can carb plates be? 11-7-12