Talk:Team:IvyTech-South Bend/19 October 2010

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== B-Galactosidase Activity Assay -- Marian Price-Carter, 9/7/00 ==
 
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Modified From Protocols Online 10/15/10
 
-
(htt p://rothlab.ucdavis.ed u/proto cols/beta-galactosidase-3 .html)
 
-
Day 1: Start overnight lactose-free broth culture.
+
== 10/19/10 ==
-
Negative control: cells lacking 13-galactasidase, such as Agrobacterium positive control: cells
+
-
with high enzyme activity E.coli.
+
-
Day 2: Dilute cells 1/100 in fresh medium now containing lactose, grow to mid-log.1 Prepare
+
Today we are doing large scale DNA extractions from E-Coli Recovering t9002 and lac-z
-
solutions: Z buffer, phosphate buffer, ONPG2.
+
-
Preparation of Cells
+
-
Incubate cultures 20’ on ice to stop grnwth and wash:
+
first taking the OD of each tube
-
¯ Pellet at least 2 mL of cells at 4 C by centrifuging 10’ at 6,000 rpm in a Sorval SS34
+
after Blacking spectrometer I will run a sample of each tube.
-
rotor.
+
-
¯ Pour off the supematant.
 
-
¯ Resuspend the cell pellet in the same volume of chilled Z buffer.
+
E-coli w/T9002 from 10/15/10 has an OD of .355 @600um
-
¯ Measure the ODr0o of the resuspended cells (blank against Z buffer)
+
E-Coli w/LacZ from 10/15/10 has an OD of 3.49 @ 600um
-
Dilute cells in Z buffer to I mL (most easily done with a pippeter). For most activities, 0.5 mL
+
Streaked E-coli w/T9002 from 10/12/10 has an OD of .363 @600um
-
cells + 0.5 mL Z buffer will produce a desirable amount of yellow color in !-2 hours. For higher
+
-
levels (>500 Miller units), try 0. I mL cells + 0.9 mL Z buffer.
+
-
Permeabilize the diluted cells by adding 100 lal chloroform and 50 ~1 0.1% SDS (sodium
+
#2 Streaked E-Coli w/LacZ from 10/15/10 has an OD of 3.26 @ 600um
-
dodecyl sulfate, sodium laurel sulfate). Chloroform is easier to pippete if the air in the pippete tip
+
-
is saturated by dm~ving up and releasing chloroform several times.
+
-
Vortex; equilibrate the tubes 5’ in a 28 C water bath.
+
-
Assay
 
-
Start reaction by adding 0.2 mL substrate, o-nitrophenyl-13-D-galactoside (ONPG; 4 mg/mL)
+
after I took 4 bottles from spec to hood and removed 5ml from each tube placing into 50 cc tubes with LB/Amp and then placed them int 37C inculbator
-
¯ Vortex - Record the time of addition precisely with timer or stopwatch.
+
Then placed the 4 tubes extracted from and placed them into centrefuge to spin down to extract DNA.
-
¯ Incubate the cells at 28 C.
+
Then following protocol from pg 19. After extracting the DNA I placed into fridge in back Room @ 4C.
-
¯ Stop the reaction after sufficient yellow color has developed3 by adding 0.5 mL 1M
 
-
Na2CO34.
 
-
¯ Vortex. - Note time of addition precisely.
+
Then made LB Broth in 3 1Lt flasks weighing 10 g in each LB mix then adding 500 ml to it, placed into microwave one time on beverage setting then into autoclave on liquid setting 121 C  @ sw
-
¯ Transfer 1 mL to an eppendorftube, spin 5’ at maximum to remove debris and
+
jhull
-
chloroform.
+
-
¯ Record the optical density at 420 nm and at 550 nm for each tube. 5
 
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¯ Calculate the units of activity6"
+
==10/19/10==
 +
I followed the protocol below
-
This is basically the assay described by J.H. Miller in "Experiments in Molecular Genetics" 1972
+
I changed my pipette tip each time to prevent cross contamination
-
Cold Spring Harbor Laboratories pages 352-355, with an extra step added. In the assay described
+
-
here, the cells are pelleted and resuspended in assay buffer (Z buffer) to eliminate error due to
+
-
the effects of different carbon sources in the growth medium on the [3-galactosidase enzyme
+
-
activity.
+
-
B-Galactosidase is able to hydrolyze (cleave) ~-D-galactosides. This enzyme facilitates growth
 
-
on carbon sources like lactose by cleaving it into a molecule of glucose and a molecule of
 
-
galactose which the cells can catabolize and grow on. In the assay described above, the substrate
 
-
o-nitrophenyl-I]-D-galactopyraniside (ONPG) is used in place of lactose. When the 13-
 
-
galactosidase cleaves ONPG, o-nitrophenol is released. This compound has a yellow color, and
 
-
absorbs 420 nm light. To measure ~-galactosidase activity the accumulation of yellow color
 
-
(increase’420 nm absorbance)/minute is monitored.
 
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Footnotes
+
==IGEM Protocols/Restriction Digest==
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~ In Sahnonella (which is naturally l]-galactosidase minus) this assay is used to monitor
+
From partsregistry.org
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transcription from insertion elements (that encode the [~-galactosidase enzyme) that have inserted
+
At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.
-
into different genes. The assay is usually performed on cells in the mid-log phase of growth. On
+
-
rich carbon sources like glucose, the OD6oo of a culture of wild-type Salmonella in mid-log phase
+
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ranges from 0.28-0.7. On poorer carbon sources or in strains that have mutations in genes that
+
-
are important for groxvth, the OD6oo at mid-log phase may be lower, since the cells may enter
+
-
stationary phase at a lower density. Therefore, before doing the assay, it is important to follow
+
-
the growth of the strain of interest in each type of medium that will be used, plot a groxvth curve,
+
-
and determine when the cells are in mid-log phase in that particular medium.
+
-
Solutions for I]-galactosidase assays
+
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Z buffer, per 50 mL:
+
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¯ O.80g Na2HPO.~.7H20 (0.06M)
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===Materials===
 +
*PCR tube
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¯ 0.28g NaH2PO4.H20 (0.04M)
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*dH20
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¯ 0.5 mL 1M KC1 (0.01M)
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*Enzymes (EcoRI, XbaI, SpeI, PstI)
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¯ 0.05 mL IM MgSO~ (0.001M)
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*BSA
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¯ 0.135 mL iB -mercaptoethanol (BME) (0.05M)
 
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¯ bring to approximately 40 mL with H20, dissolve all the salts
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*Enzyme Buffer (NEBuffer 2)*
 +
Notes: You should keep all materials on ice.
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¯ adjust the pH to 7.0
 
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¯ use a graduated cylinder to bring the buffer to 50 mL
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===Protocol===
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¯ store at4 C.
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1.  Add 500n~ of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
 +
2.  Add 5ul ofNEBuffer 2 to the tube.
-
Note: BME is added to the reaction buffer to stabilize the ~-galactosidase enzyme. The important
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3. Add 0.5ul of BSA to the robe.
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part of BME is a reactive thiol (SH group). Thiols react with oxygen in the air and oxidize
+
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(inactivate) over time. Therefore, try not to make much more Z buffer than you will use in a few
+
-
days. Store the unused portion at 4 C.
+
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ONPG should be dissolved fresh each day. Dissolve 1.5X as much as you think you will need,
+
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because you may have to repeat one or more of the assays Le. for a different amount of time or
+
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with a different cell dilution. Dissolve the ONPG to a final concentration of 4mg/mL in 0. IM
+
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phosphate buffer pH 7.0.
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4. Add l ul of your first enzyme
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Phosphate buffer, per 100 mL:
+
5.  Add l ul ofyour second e~e.(~¢,
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¯ 1.61g Na2HPO4.7H20 (0.06M)
+
6. There should be a total volume of 5(iul. Mix well and spin down.
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¯ 0.55g NaH2PO4.H~O (0.04M)
+
7. Incubate the restriction digest at_3_7C for 30miri, and then 80C for 20min to heat kill the enzymes.
 +
l~e incubate in a thermocycler with a heat~-d lid
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¯ adjust the pH to 7.0
+
8.  Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2u of the digest (20mg of DNA) for Ligations.
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¯ phosphate buffer is stable at room temperature and does not need to be made fresh each
+
===New restriction and Ligation===
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time.
+
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3 What is sufficient yellow color? To get the most accurate measure of activity, the absorbance at
+
Joe and I perfomed new restriction and ligation
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420 nm (A42o) should range.from 0.6 to 0.9. Readings as low as 0.1 and as high as 1.2 are
+
We did the sam protocol for the first restriction and ligation
-
acceptable. Tubes that have become as yellow as a tube of(unused) LB broth will probably be
+
-
sufficiently yellow.
+
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If the reading is too low, try the assay again with more cells or longer incubation time. When the
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The DNA was put into thermocycler 30 min @ 37C and 20 min @ 80C
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element has inserted into a gene that is not expressed much, it will probably take hours to
+
-
develop enough yellow color. If your negative control starts to tum yellow (after several or more
+
-
hours) it means that the substrate is beginning to auto-hydrolyze. The assay can be left overnight.
+
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The auto-hydrolysis is then accounted for by subtracting the A42o and A~oo of the negative control
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Stored @ 4.0C in thermocycler
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from that of the tests before doing any further calculations.
+
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If the reading is too high, try the assay again with fewer cells¯ Aim to stop the reaction after 15
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*unsure of the date
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minutes. For example, if in your first attempt, you added 0.5 mL 0fcells + 0.5 mL of Z buffer,
+
*will be used for electrophoresis
-
and it was too yellow after 5 minutes, try adding 0.1 mL cells + 0.9 mL of Z buffer. Watch the
+
-
tube carefully. Some cultures may have to be diluted even further[
+
-
~ Adding the 1 M Na~CO3 stops the reaction by raising the pH of the solution to 11. At this pH
 
-
the enzyme is not active.
 
-
5 The reading at 420 nm is a combination of absorbance by o-nitrophenol and light scattering by
+
==Using electrocom cells/DNA for electroporateion==
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cell debris. The absorbance at 550 corrects for light scattering. There is no absorbance from onitrophenol
+
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at this wavelength. The light scattering at 420 nm is proportional to that at 550 rim:
+
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light scattering at 420 nm = 1.75 x OD55o
+
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6 Use the following equation to calculate units of enzyme activity:
+
Procedure completed as follows
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Miller Units = I000 x [(OD~o - 1.75 x OD~5o)] / (T x V x OD6~o)
+
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¯ OD~o and OD~o am mad from the reaction mixture.
+
1. Place 1-5 btl of DNA in a sterile microcentrifuge tube and c[qill on ice. For the
 +
pUCl9 control DNA, use l ul (10pg.) (l ul used)
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¯ OD6oo reflects cell density in the washed cell suspension.
+
Caution: The DNA must be resuspended in water (rather than TE buffer) to keep
 +
the ionic strength to a minimum. Otherwise, arcing might occur during the pulse,
 +
which can damage the machine and will result in no transformation and cell death.
 +
DNA in ligation or restriction buffer must be precipitated or desalted before
 +
electroporation.
-
¯ T = time of the reaction in minutes¯
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2. Gently thaw cells on ice. Use these cells immediately, do not leave them on ice for
 +
an extended period of time. Unused cells can be re-frozen for later use, but they will
 +
suffer a significant loss of efficiency.
-
¯ V = volume of culture used in the assay in mLs.
+
3. Transfer the desired amount of cells to the pre-chilled microcentrifuge tube
 +
containing the DNA. It is recommended that 20-40 ~tl (40-80 ~1 if using 0.2 cm
 +
cuvettes) be used for a small-scale electroporation (< 100 ng of DNA), and 160 ul
 +
for a large-scale or library electroporation. The volume of DNA added should not
 +
exceed 5% of the total cell/DNA mixture. Mix the cells gently. Do not mix by
 +
pipetting up and down. Leave the cells on ice for 1 minute.
-
The units give the change in A4~o/min/mL of cells/OD6oo
+
4. After 1 minute, transfer the cells to a chilled cuvette and gently shake them to the
 +
bottom of the cuvette. Check the cuvette by looking at the contents from both sides
 +
to make sure the cells make contact all the way across the bottom of the chamber
 +
without any air bubbles. Do this as quickly as possible, being careful not to warm up
 +
the cuvette and cells. Remove the condensation from the outside surfaces of the
 +
cuvette with a tissue.
 +
5. Electroporate your sample using the settings listed on the previous page or the
 +
manufacturer’s recommended settings.
-
Typical values:
+
6. Immediately add 480 ~tl (for small-scale) or 960 btl (for large-scale) of SOC medium
 +
to the cells.
 +
7. Transfer the suspension to a 15 ml tube. Incubate it at 37°C in a rotary shaking
 +
incubator at 225 rpm for 1 hour to allow expression of the antibiotic resistance.
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a fully induced lac+ operon (+IPTG) = 1500 units
+
8.  Plate the cells on prewarmed selective plates and incubate overnight at 37°C. We
 +
recommend plating 2 different volumes to ensure that at least 1 plate has wellspaced
 +
colonies.
-
an uniduced lac÷ operon (no IPTG) = 1.5-3 units
+
Note: For the pUC19 control DNA, make a 1:10 dilution in SOC medium and plate --’-
 +
_ 25/ul and 100 ul of cells on LB agar with 100 ug/ml of ampicillin.
 +
 
 +
 
 +
=== Tubes labeled A-D ===
 +
 
 +
 
 +
I’m beginning w/Tube A (DNA incubated)
 +
 
 +
180 kv - 4.5 ms for tube A
 +
 
 +
 
 +
Tube B
 +
 
 +
same protocol as A
 +
 
 +
180 kv – 3.4ms for tube B
 +
 
 +
used 2 ul of DNA instead of 1
 +
 
 +
put into 37C for incubation
 +
 
 +
Tub C
 +
same protocol as A
 +
 
 +
180 kv – 3.5ms for tube C
 +
 
 +
used 2 ul of DNA instead of 1
 +
 
 +
put into 37C for incubation
 +
 
 +
Tube D
 +
same protocol as A
 +
 
 +
180 kv – 4.3 ms for tubeD
 +
 
 +
used 2 ul of DNA instead of 1
 +
 
 +
put into 37C for incubation
 +
 
 +
Plating DNA/Cells
 +
This will be done for tube A-D
 +
 
 +
Using Sterile pre-poured plates
 +
 
 +
add 100ul of DNA/Elec.Com/SOB media
 +
 
 +
Spread then incubate
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
dgarvey

Latest revision as of 18:49, 27 October 2010


Contents

10/19/10

Today we are doing large scale DNA extractions from E-Coli Recovering t9002 and lac-z

first taking the OD of each tube

after Blacking spectrometer I will run a sample of each tube.


E-coli w/T9002 from 10/15/10 has an OD of .355 @600um

E-Coli w/LacZ from 10/15/10 has an OD of 3.49 @ 600um

Streaked E-coli w/T9002 from 10/12/10 has an OD of .363 @600um

  1. 2 Streaked E-Coli w/LacZ from 10/15/10 has an OD of 3.26 @ 600um


after I took 4 bottles from spec to hood and removed 5ml from each tube placing into 50 cc tubes with LB/Amp and then placed them int 37C inculbator

Then placed the 4 tubes extracted from and placed them into centrefuge to spin down to extract DNA.

Then following protocol from pg 19. After extracting the DNA I placed into fridge in back Room @ 4C.


Then made LB Broth in 3 1Lt flasks weighing 10 g in each LB mix then adding 500 ml to it, placed into microwave one time on beverage setting then into autoclave on liquid setting 121 C @ sw

jhull


10/19/10

I followed the protocol below

I changed my pipette tip each time to prevent cross contamination


IGEM Protocols/Restriction Digest

From partsregistry.org At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.

Materials

  • PCR tube
  • dH20
  • Enzymes (EcoRI, XbaI, SpeI, PstI)
  • BSA


  • Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.


Protocol

1. Add 500n~ of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.

2. Add 5ul ofNEBuffer 2 to the tube.

3. Add 0.5ul of BSA to the robe.

4. Add l ul of your first enzyme

5. Add l ul ofyour second e~e.(~¢,

6. There should be a total volume of 5(iul. Mix well and spin down.

7. Incubate the restriction digest at_3_7C for 30miri, and then 80C for 20min to heat kill the enzymes. l~e incubate in a thermocycler with a heat~-d lid

8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2u of the digest (20mg of DNA) for Ligations.

New restriction and Ligation

Joe and I perfomed new restriction and ligation

We did the sam protocol for the first restriction and ligation

The DNA was put into thermocycler 30 min @ 37C and 20 min @ 80C

Stored @ 4.0C in thermocycler

  • unsure of the date
  • will be used for electrophoresis


Using electrocom cells/DNA for electroporateion

Procedure completed as follows

1. Place 1-5 btl of DNA in a sterile microcentrifuge tube and c[qill on ice. For the pUCl9 control DNA, use l ul (10pg.) (l ul used)

Caution: The DNA must be resuspended in water (rather than TE buffer) to keep the ionic strength to a minimum. Otherwise, arcing might occur during the pulse, which can damage the machine and will result in no transformation and cell death. DNA in ligation or restriction buffer must be precipitated or desalted before electroporation.

2. Gently thaw cells on ice. Use these cells immediately, do not leave them on ice for an extended period of time. Unused cells can be re-frozen for later use, but they will suffer a significant loss of efficiency.

3. Transfer the desired amount of cells to the pre-chilled microcentrifuge tube containing the DNA. It is recommended that 20-40 ~tl (40-80 ~1 if using 0.2 cm cuvettes) be used for a small-scale electroporation (< 100 ng of DNA), and 160 ul for a large-scale or library electroporation. The volume of DNA added should not exceed 5% of the total cell/DNA mixture. Mix the cells gently. Do not mix by pipetting up and down. Leave the cells on ice for 1 minute.

4. After 1 minute, transfer the cells to a chilled cuvette and gently shake them to the bottom of the cuvette. Check the cuvette by looking at the contents from both sides to make sure the cells make contact all the way across the bottom of the chamber without any air bubbles. Do this as quickly as possible, being careful not to warm up the cuvette and cells. Remove the condensation from the outside surfaces of the cuvette with a tissue.

5. Electroporate your sample using the settings listed on the previous page or the manufacturer’s recommended settings.

6. Immediately add 480 ~tl (for small-scale) or 960 btl (for large-scale) of SOC medium to the cells. 7. Transfer the suspension to a 15 ml tube. Incubate it at 37°C in a rotary shaking incubator at 225 rpm for 1 hour to allow expression of the antibiotic resistance.

8. Plate the cells on prewarmed selective plates and incubate overnight at 37°C. We recommend plating 2 different volumes to ensure that at least 1 plate has wellspaced colonies.

Note: For the pUC19 control DNA, make a 1:10 dilution in SOC medium and plate --’- _ 25/ul and 100 ul of cells on LB agar with 100 ug/ml of ampicillin.


Tubes labeled A-D

I’m beginning w/Tube A (DNA incubated)

180 kv - 4.5 ms for tube A


Tube B

same protocol as A

180 kv – 3.4ms for tube B

used 2 ul of DNA instead of 1

put into 37C for incubation

Tub C same protocol as A

180 kv – 3.5ms for tube C

used 2 ul of DNA instead of 1

put into 37C for incubation

Tube D same protocol as A

180 kv – 4.3 ms for tubeD

used 2 ul of DNA instead of 1

put into 37C for incubation

Plating DNA/Cells This will be done for tube A-D

Using Sterile pre-poured plates

add 100ul of DNA/Elec.Com/SOB media

Spread then incubate




dgarvey

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