Talk:Team:IvyTech-South Bend/8 October 2010

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(→Electroporation)
 
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== 1:00 ==
== 1:00 ==
 +
 +
Now I'm going to  electroporate staph electocomp cells wtih part T9002 from NDA extraction done by HY.
 +
Following protocol below:
 +
 +
=== Electroporation ===
 +
 +
1. Pipette the DNA samples (5 ng- 2 lag in a volume - 3 lal) to be electroporated into
 +
sterile 1.5 ml microfuge tubes.
 +
 +
Thaw the competent cells at room temperature for several minutes. Add 50 ~tl of cells to
 +
each DNA sample; gently pipette up and down to mix.
 +
 +
Incubate the samples at room temperature for 30 min.
 +
 +
Set the MicroPulser to "StA". See Section 4 for operating instructions.
 +
 +
Transfer the mixture of cells and DNA to a 0.2 cm electroporation cuvette and tap the
 +
suspension to the bottom &the tube.
 +
Place the cuvette in the chamber slide. Push the
 +
slide into the chamber until the cuvette is seated between the contacts in the base of the
 +
chamber.
 +
 +
Pulse once.
 +
 +
Remove the cuvette from the chamber and immediately add 1 ml of SMMP medium
 +
containing a subinhibitory concentration of antibiotic; gently transfer the cells to a sterile
 +
17 x 100 mm tube using a Pasteur pipette. Incubate 1 hr at 37 °C, shaking at 250 rpm.
 +
Check and record the pulse parameters. The time constant should be close to
 +
2.5 milliseconds.
 +
 +
The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
 +
 +
Plate aliquots of the electroporated cells on trypticase soy agar containing selective antibiotic.
 +
 +
Incubate plates for 36-48 hrs at 37 °C.
 +
 +
 +
 +
subinhibitory concentration of antibiotic?
 +
guessed at 1:100 dilution further than normal so 10 ul of SMMP = 10(4) ul of SMMP
 +
 +
need 0.1 ul of out 1000x Amp stock
 +
 +
Pulse parameter 1.79 kv 2.50 ms (good!)
 +
 +
--JHull
 +
 +
 +
 +
=== NO GROWTH ON LB ===
 +
 +
No agrowth apparent on LB
 +
Set up Broth Culture in TSB 4% (40g/1L) 6/13/10 AMR.
 +
 +
25 mls + 25 ulll AMP (from J4) + 500 ul tranf. mix
 +
 +
I13522 transformed E-Coli from 10/7/10 is showing growth and good florescence. Picked one colony and transferred to LB AMP broth on Shaker 23C.

Latest revision as of 20:55, 17 October 2010

Contents

10/8/10

Making 4 1L bottlse of LB-Lennox Broth

2- with Agar 2- without

I placed between 10.035 - 10.005 grams into each of the 4 bottles, then added 500 ml 18 ohm H2O into each bottle

Placed into the microwave on beverage setting 8oz.

after first time removed the 2 -just broth

heated the 2- with Agar again at the same setting.

Added to 2- with Agar - 7.002 grams to each then placed into the autoclave on liquid setting/ 121 C for 15 min.

After done , cool to room temp label and place into fridge until needed.


1:00

Now I'm going to electroporate staph electocomp cells wtih part T9002 from NDA extraction done by HY. Following protocol below:

Electroporation

1. Pipette the DNA samples (5 ng- 2 lag in a volume - 3 lal) to be electroporated into sterile 1.5 ml microfuge tubes.

Thaw the competent cells at room temperature for several minutes. Add 50 ~tl of cells to each DNA sample; gently pipette up and down to mix.

Incubate the samples at room temperature for 30 min.

Set the MicroPulser to "StA". See Section 4 for operating instructions.

Transfer the mixture of cells and DNA to a 0.2 cm electroporation cuvette and tap the suspension to the bottom &the tube. Place the cuvette in the chamber slide. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.

Pulse once.

Remove the cuvette from the chamber and immediately add 1 ml of SMMP medium containing a subinhibitory concentration of antibiotic; gently transfer the cells to a sterile 17 x 100 mm tube using a Pasteur pipette. Incubate 1 hr at 37 °C, shaking at 250 rpm. Check and record the pulse parameters. The time constant should be close to 2.5 milliseconds.

The field strength can be calculated as actual volts (kV) / cuvette gap (cm).

Plate aliquots of the electroporated cells on trypticase soy agar containing selective antibiotic.

Incubate plates for 36-48 hrs at 37 °C.


subinhibitory concentration of antibiotic? guessed at 1:100 dilution further than normal so 10 ul of SMMP = 10(4) ul of SMMP

need 0.1 ul of out 1000x Amp stock

Pulse parameter 1.79 kv 2.50 ms (good!)

--JHull


NO GROWTH ON LB

No agrowth apparent on LB Set up Broth Culture in TSB 4% (40g/1L) 6/13/10 AMR.

25 mls + 25 ulll AMP (from J4) + 500 ul tranf. mix

I13522 transformed E-Coli from 10/7/10 is showing growth and good florescence. Picked one colony and transferred to LB AMP broth on Shaker 23C.

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