Team:IPN-UNAM-Mexico/Protocols

=Protocols=

MIDIPREP
Modified from “QIagen Plasmid Purification Midi”.


 * 1) Inoculate a colony in 25 - 50 ml of LB Medium with antibiotic. For low copy plasmids, inoculate 100 - 200 ml of medium.
 * 2) Centrifuge 20 minutes at 4000 rpm and get the pellet.
 * 3) Suspend the pellet in 4 ml of P1 QIagen Buffer (Be sure to add RNAse to the buffer).
 * 4) Add 4 ml of P2 QIagen and mix on inversion 6 times.
 * 5) Add 4 ml of P3 QIagen and mix on inversion, add 10 ml of chloroform and mix on inversion 6 times.
 * 6) Incubate on ice 10 minutes and centrifugate 20 minutes at 4000 rpm.
 * 7) Recover the aqueous phase and add 5ml of isopropanol, incubate 30 minutes on ice.
 * 8) Centrifugate 15 minutes at 13000 rpm.
 * 9) 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

 * 1) Take 5 ml of liquid SOB and incubate at 37 °C overnight
 * 2) 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
 * 3) Transfer the cells to 250 ml bottles (must be cold)
 * 4) Incubate for 30 minutes on ice
 * 5) Centrifuge at 2500 rpm for 15 min at 4°C.
 * 6) Resuspend cells in 20 ml of ice-cold RF1 solution
 * 7) Incubate for 15 min on ice
 * 8) Centrifuge at 2500 rpm for 15 min at 4°C.
 * 9) Resuspend cells in 2 ml of ice-cold RF2 solution.
 * 10) 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 %


 * 1) 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 %


 * 1) Adjust pH to 6.8 with 0.2 M of NaOH and sterilize by filtration using a 0.22 μm filter

CTAB miniprep

 * 1) Take 1.5 ml of cell culture and centrifuge at 15000 rpm for 3 minutes.
 * 2) Discard liquid phase
 * 3) Repeat 1 and 2
 * 4) Resuspend cells with 1 ml of NaCl 1.2 M with vortex
 * 5) Centrifuge at 15000 rpm for 3 minutes and discard supernatant.
 * 6) Add 100 μl of sterile water and resuspend with vortex
 * 7) Add 200 μl of STET and mix with vortex.
 * 8) Add 4 μl of lysozyme and mix with vortex.
 * 9) Incubate for reaction to occur for 5 min at 37°C then boil for 45 seconds.
 * 10) Boil 45 seconds inside a glass with water
 * 11) Centrifuge at 15000 rpm for 10 min.
 * 12) Discard pellet.
 * 13) Add 8 μl of CTAB and centrifuge at 15000 rpm for 5 min.
 * 14) Discard supernatant
 * 15) Resuspend with 300 μl of NaCl 1.2 M using vortex.
 * 16) Add 1 ml of ethanol and incubate at -20°C for 20 min.
 * 17) Centrifuge at 15000 rpm for 10 min.
 * 18) Discard supernatant.
 * 19) Wash with 1 ml of 70% ethanol and centrifuge for 3 min.
 * 20) Drop out ethanol by decantation.
 * 21) To dry remaining ethanol use a thermoblock at 65°C.
 * 22) Resuspend with 100 μl of sterile water and add 1 μl of RNase.
 * 23) Incubate at 37°C for 30 min.

DNA purification from agarose gel

 * 1) In a UV room, cut the gel with a knife and place DNA bands inside falcon tubes
 * 2) Place tubes on a tared balance to get the gel weight.
 * 3) Multiply the gel weight by three to obtain the volume of QG buffer to be added
 * 4) Add the volume of QG buffer obtained in the previous step.
 * 5) Place tubes on thermoblock for 10 min at 55°C.
 * 6) Prepare one column for every tube.
 * 7) Take 800 μl of the tubes and place the volume in the column.
 * 8) Centrifuge the columns at 13000 rpm for 1 minute with the lid open.
 * 9) Add 800 μl of PE to every column and wait 6 minutes.
 * 10) Centrifuge at 13000 rpm for 1 minute.
 * 11) Discard supernatant and centrifuge again at 13000 rpm for 30 seconds.
 * 12) Place column inside a Eppendorf tube
 * 13) Add 25 μl of deionized water to the column and wait for 6 minutes.
 * 14) Centrifuge at 15000 rpm for 2 minutes.