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Preparation of competent cells
1. Grow overnight in LB
2. Add 1ml of culture to 100ml of TY. Or SOC. Or LB.
3. Grow until it reaches an O.D. of 0.4
4. Place at 4C for 10 minutes
5. Centrifuge for 20 min. at 3000 rpm.
6. Decant the supernatant and add 10 ml of 100mM MgCl2 and resuspend
7. Spin for 10 minutes at 3,000 rpms. Decant supernatant
8. Resuspend in 50mL of 100mM CaCl2
9. Leave at 4C for 20 minutes
10. Spin for 20 minutes at 30,000 rpm
11. Resuspend in 10 mL of 100mM CaCl2 and 10% Glycerol
12. Aliquot by 150uL ( 2x )
13. Freeze in liquid Nitrogen and store at -80
Thermal Transformation
1. Add 2uL of Ligation mixture to TOP10'F one shot comp cells
2. Incubate on ice for about 5 minutes
3. Heat Shock the cells for 30 seconds at 42oC
4. Transfer tubes to ice and immediately add 250 uL of SOC.
5. Cap the tubes and horizontally shake at 37oC for 30 min.
6. Spread 50uL of the cells upon LB/Amp plates
7. Incubate at 37oC overnight
Miniprep of DNA vectors with QIAGEN kits
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PCR to Determine if Insert is Present and in Correct Orientation by
PCR
mix for 5 reactions :
5uL of T7promoter forward primer (5pmol/uL)
5uL of anticoding primer (100nmol/uL)
40uL of PCR supermix Gibco-BRL
add 10uL of this mix to 1uL of miniprep DNA.
PCR 5min 95oC, 30 sec. 95oC, 30 sec. 58oC, 25-35 cycles 1min 72oC, 10min
72oC, hold at 4oC
Sequencing by PCR
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Testing for Expression
1. Transform plasmids having gene of interest and immediately inoculate
a 4ml culture. -LB having 100umol of Amp and 34umol chlorophenicol -grow
overnight.
2. Inoculate 50mL of LB with 500uL of overnight cultures (be sure to add
all of the proper antibiotics to media)
3. Grow the cells until they reach an OD600 of 0.5 to 0.8
4. Take 1mL from each sample, then add 50uL of 1mM IPTG. Keep the samples
at room temp.
5. Grow cells for 1 hr, then take another 1mL sample and keep at room
temp.
6. Grow cells for another 2 hrs and then take a 1mL sample from each culture.
7. Spin the 1mL samples down and aspirate the supernatant.
8. Compare the pellet sizes of the first, second and third rounds of samples
and Add 20mM Tris pH 7.5 accordingly. Example: If the initial pellet before
induction is only half the size as the samples from the 3rd round of samples
then add 40uL of Tris while adding exactly double the amount to the 3rd
round of samples. This insures that the samples will be of about the same
concentration when running on the gel.
9. Make sure that Tris is added first and are vortexed before the addition
of a same volume as Tris buffer of 2X Loading SDS-Buffer ( add 2uL of
BME to reduce cystein bonds ). Vortex
10. Boil the samples at 100oC for 1 minutes. Centrifuge briefly to bring
down condensation.
11. Vortex the samples and make sure that the samples are easily drawn
into a loading pipet tip. If not then break up the lysate by drawing through
a syringe. Note: make sure you keep the 50mL cultures around so that you
can perform protein purification On those that are positive for protein
expression.
Expression of cytoplasmic proteins
1. Inoculate a 4ml culture. -LB having 100umol of Amp and 34umol chlorophenicol
-grow overnight.
2. Inoculate 500ml of LB containing 100umol/ml Amp and 34umol/ml chloramphenicol
with 1ml of your overnight culture.
3. Grow at 37oC untill the OD is at 0.5-0.8
4. Add 500 ul of 1M IPTG (1mM final) . Grow for 3-6 hours
5. Spin down to pellet the E.coli.
6. Wash cells 2 times with Tris 20mM, EDTA 1mM, pH 7.5, 4oC, resuspend,
vortex and immediately centrifuge at 4oC
7. Resuspend in 10mL of 20mM Tris, EDTA 0.5mM pH 7.5 50uL
8. Lysate these samples by sonication.
9. Centrifuge, discart the pellet.
10. Check the protein expression on SDS-PAGE
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Experimental
protocols
1- Gene cloning