From 2009.igem.org

Revision as of 03:51, 22 October 2009

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.

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