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Team:IPN-UNAM-Mexico/Protocols
From 2009.igem.org
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MIDIPREP
Francisco Razo Hernández (noviembre 2008). Modified from “QIagen Plasmid Purification Midi”.
- Inoculate a colony in 25 - 50 ml of LB Medium with antibiotic. For low copy plasmids, inoculate 100 - 200 ml of medium.
- Centrifuge 20 minutes at 4000 rpm and get the pellet.
- Suspend the pellet in 4 ml of P1 QIagen Buffer (Be sure to add RNAse to the buffer).
- Add 4 ml of P2 QIagen and mix on inversion 6 times.
- Add 4 ml of P3 QIagen and mix on inversion, add 10 ml of chloroform and mix on inversion 6 times.
- Incubate on ice 10 minutes and centrifugate 20 minutes at 4000 rpm.
- Recover the aqueous phase and add 5ml of isopropanol, incubate 30 minutes on ice.
- Centrifugate 15 minutes at 13000 rpm.
- Wash with EtOH at 70% and centrifugate 2 min at 13000 rpm, let the pellet dry and suspend on 500 ml of esterile deionized water (Water for pcr).
Competent cells with RbCl
- Take 5 ml of liquid SOB and incubate at 37 °C overnight
- Take 1 ml of cell culture and innoculate into 500 ml of YENB media and incubate at 37 °C and 200 rpm until O.D.=0.5-0.55
- Transfer the cells to 250 ml bottles (must be cold)
- Incubate for 30 minutes on ice
- Centrifuge at 2500 rpm for 15 min at 4°C.
- Resuspend cells in 20 ml of ice-cold RF1 solution
- Incubate for 15 min on ice
- Centrifuge at 2500 rpm for 15 min at 4°C.
- Resuspend cells in 2 ml of ice-cold RF2 solution.
- Incubate for 15 min on ice and divide into 200 μl aliquots and store at -70°C
RF1 solution
- Rubidium chloride....................... 100 mM
- Manganese chloride tetrahydrate......... 50 mM
- Potassium Acetate....................... 30 mM
- Calcium chloride dihydrate.............. 10 mM
- Gycerol................................. 15 %
- Adjust pH to 5.8 with 0.2 M of glacial acetic acid and sterilize by filtration using a 0.22 μm filter
RF2 solution
- Rubidium chloride....................... 100 mM
- MOPS.................................... 10 mM
- Calcium cloride dihydrate............... 75 mM
- Gycerol................................. 15 %
- Adjust pH to 6.8 with 0.2 M of NaOH and sterilize by filtration using a 0.22 μm filter
CTAB miniprep
- Take 1.5 ml of cell culture and centrifuge at 15000 rpm for 3 minutes.
- Discard liquid phase
- Repeat 1 and 2
- Resuspend cells with 1 ml of NaCl 1.2 M with vortex
- Centrifuge at 15000 rpm for 3 minutes and discard supernatant.
- Add 100 μl of sterile water and resuspend with vortex
- Add 200 μl of STET and mix with vortex.
- Add 4 μl of lysozyme and mix with vortex.
- Incubate for reaction to occur for 5 min at 37°C then boil for 45 seconds.
- Boil 45 seconds inside a glass with water
- Centrifuge at 15000 rpm for 10 min.
- Discard pellet.
- Add 8 μl of CTAB and centrifuge at 15000 rpm for 5 min.
- Discard supernatant
- Resuspend with 300 μl of NaCl 1.2 M using vortex.
- Add 1 ml of ethanol and incubate at -20°C for 20 min.
- Centrifuge at 15000 rpm for 10 min.
- Discard supernatant.
- Wash with 1 ml of 70% ethanol and centrifuge for 3 min.
- Drop out ethanol by decantation.
- To dry remaining ethanol use a thermoblock at 65°C.
- Resuspend with 100 μl of sterile water and add 1 μl of RNase.
- Incubate at 37°C for 30 min.
DNA purification from agarose gel
- In a UV room, cut the gel with a knife and place DNA bands inside falcon tubes
- Place tubes on a tared balance to get the gel weight.
- Multiply the gel weight by three to obtain the volume of QG buffer to be added
- Add the volume of QG buffer obtained in the previous step.
- Place tubes on thermoblock for 10 min at 55°C.
- Prepare one column for every tube.
- Take 800 μl of the tubes and place the volume in the column.
- Centrifuge the columns at 13000 rpm for 1 minute with the lid open.
- Add 800 μl of PE to every column and wait 6 minutes.
- Centrifuge at 13000 rpm for 1 minute.
- Discard supernatant and centrifuge again at 13000 rpm for 30 seconds.
- Place column inside a Eppendorf tube
- Add 25 μl of deionized water to the column and wait for 6 minutes.
- Centrifuge at 15000 rpm for 2 minutes.
