Latest revision as of 21:05, 25 June 2012

Protocols

LB plates with antibiotics Protocol:

Follow the recipe card in box for making LB plates, being sure to add the agar. After autoclaving, and when the agar has cooled enough that it’s not too hot to touch (about 1 to 1.5hrs), add antibiotics as follows:

Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plate with a single red line on the side.
Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml. Mark the plates with a single green line on the side.
Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml. Mark the plates with a single black line on the side.
Chloramphenicol – add 1ml chloramphenicol stock (at 34mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plates with a single purple line on the side

Arabinose: 15mg/mL = 100x stock

Plain LB Plate Protocol:

(500 ml) LB plates: In a 1 liter flask place 2.5 grams of yeast extract, 5 grams Bacto-Tryptone, 5 grams sodium chloride and 7.5 grams of agar.
Add distilled water to the mixture until the volume is about 500 milliliters.
Cover the top of the flask with foil and autoclave the solution for 20 minutes at 121 degrees Celsius (250 degrees Fahrenheit).
Remove the flask from the autoclave and let cool until the bottom of the flask is warm to the touch.
Pour the solution into a petri dish until about 1/3 full.
Loosely replace the lid on the dish and let cool until condensation stops.

Source: http://biology.about.com/c/ht/00/07/How_LB_For_Bacterial0962932482.htm

Transformation protocol from Lucigen (Ecloni)

1. Chill sterile culture tubes on ice (17 mm x 100 mm tubes, one tube for each transformation reaction).
2. Remove E. cloni EXPRESS cells from the -80°C freezer and thaw completely on wet ice (10-20 minutes).
3. Add 40 µl of E. cloni EXPRESS cells to the chilled culture tube.
4. Add 1 µl of ligation reaction or DNA sample to the 40 µl of cells on ice. (Failure to purify or heat inactivate the ligation reaction may prevent transformation.) Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells.
5. Incubate on ice for 30 minutes.
6. Heat shock cells by placing them in a 42 water bath for 45 seconds.
7. Return the cells to ice for 2 minutes.
8. Add 960 µl of room temperature Expression Recovery Medium to the cells in the culture tube.
9. Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37C.
10. Plate up to 100 µl of transformed cells on LB agar plates containing the appropriate antibiotic.
11. Incubate the plates overnight at 37°C.
12. Transformed clones can be further grown in LB or any other lactose minus medium.

Source: http://lucigen.com/store/docs/manuals/MA019_Ecloni%20EXPRESS_Chemically_Competent.pdf

Transformation Protocol for Invitrogen One Shot Top10 Chemically Competent Cells:

1. Thaw, on ice, one vial of One Shot®TOP10 chemically competent cells for each transformation.
2.Add 1 μl of the DNA into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1 μl) of DNA into a separate vial of One Shot® cells and mix gently.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat-shock the cells for 30 seconds at 42°C without shaking.
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
6. Aseptically add 250 μl of pre-warmed S.O.C. Medium to each vial.
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpmin a shaking incubator.
8. Spread 100 μl from each transformation on a pre-warmed selective plate and incubate overnight at 37°C. Store remaining mix at 4C.

Source: http://tools.invitrogen.com/content/sfs/manuals/oneshottop10_chemcomp_man.pdf

MiniPrep for Puc19 using Quiagen Procedure:

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30–60 s and discard the flow-through.
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB.?? Centrifuge for 30–60 s and discard the flow-through.
Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. 􀁓 Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Source:http://www.qiagen.com/literature/handbooks/literature.aspx?id=1000248

Restriction Digest Protocol

Reaction Volumes - DNA (plasmid)-500ng, Restriction Enzymes-1ul, Buffer-5ul, BSA-0.5ul, add H2O to total volume of 50ul. Incubate at 37C for 10 min and then heat inactivate at 80C for 20min.

Source: http://www.neb.com/nebecomm/products/protocol445.asp

Ligation Reaction Protocol:

1. Upstream part digestion-2ul, downstream part digestion-2ul, backbone digestion-2ul, 10x T4 DNA Ligase Buffer-2ul, T4 DNA Ligase-1ul, H2O-11ul
2. Incubate at room temp 10min, then heat inactivate at 80C for 20 min.
3. Transform 2ul ligation product into 50ul competent E. coli cells.

Source: http://www.neb.com/nebecomm/products/protocol446.asp

Sonication Protocol:

-cool cells on ice for 10 min
-Trial 1: Sonicate for 10 sec intervals on high with 30 sec on ice in between - do this 10 times
-Trial 2: Sonicate for 10 sec intervals on low with 30 sec on ice in between - do this 10 times

Source: Experimentally determined and tested

PCR Protocol for LuxBrick:

-25 ul total reaction volume
-12.5 ul Taq98 master mix (includes Taq pol, reaction buffer, dNTPs, MgCl, other buffers)
-1.25 ul F primer, 1.25 ul R primer
-I’m using 5 ul cell extract
-fill rest of volume with water

Source:
http://lucigen.com/store/docs/manuals/MA126-Taq98-Hot-Start.pdf
http://www.protocol-online.org/biology-forums/posts/23706.html
http://cdn.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf

Site-Directed Mutagenesis Protocol:

Source: http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf

Antarctic Phosphatase Protocol:

-Add 10x buffer to dilute to 1x in restriction digest reaction (after digest enzymes are inactivated)
-Add 1ul antarctic phosphatase
-Incubate 37C for 1 hr and then 65C for 5 minutes

Making and using Electrocompetent Cells:

1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm.
2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks.
3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
6. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
7. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before.
8. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
9. At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for >6 months.

Source: http://www.neb.com/nebecomm/tech_reference/competent_cells/electrocompetentcells.asp#.T-SU4c2_BnI

Electroporation:

1. Turn on electroporator and set to 2.0 kV, 200 ohms and 25 µF.
2. Place recovery SOC in 37°C water bath.
3. Pre-warm LB-antibiotic plates at 37°C.
4. Thaw cells on ice for 10 min.
5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
6. Flick the tube containing cells a few times to mix and add 50 µl to the microcentrifuge tubes.
7. Add 1-3 µl of DNA solution (in DI water) to the cells in the microcentrifuge tube.
8. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
9. Immediately add 950 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
10. Rotate in the 37°C incubator for 1 h.
11. Make appropriate dilutions in SOC and plate.
12. Incubate at 37°C for 18 h.

NT Cell Transformation Protocol

BY-2 (NT1) Cell Transformation with Agrobactrium

Day 1:

1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary.

Day 2:

2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture, which receives no bacteria.
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished.
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation.
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA.
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C.

Day 5:

7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC.
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor.
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.
11. Resuspend in 50 ml NTC and repeat spin.
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes.
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks.

@@ Line 128: / Line 128: @@
 -Add 1ul antarctic phosphatase<br>
 -Incubate 37C for 1 hr and then 65C for 5 minutes<br>
+<br>
+<b>Making and using Electrocompetent Cells: </b><br>
+<br>
+. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm.<br>
+. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. <br>
+. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells <br>
+. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles. <br>
+. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.<br>
+. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down. <br>
+. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before. <br>
+. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. <br>
+. At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for >6 months.<br>
+<br>
+Source: http://www.neb.com/nebecomm/tech_reference/competent_cells/electrocompetentcells.asp#.T-SU4c2_BnI<br>
+<br>
+<b>Electroporation:</b><br>
+<br>
+. Turn on electroporator and set to 2.0 kV, 200 ohms and 25 µF.<br>
+. Place recovery SOC in 37°C water bath.<br>
+. Pre-warm LB-antibiotic plates at 37°C.<br>
+. Thaw cells on ice for 10 min.<br>
+. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.<br>
+. Flick the tube containing cells a few times to mix and add 50 µl to the microcentrifuge tubes.<br>
+. Add 1-3 µl of DNA solution (in DI water) to the cells in the microcentrifuge tube.<br>
+. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).<br>
+. Immediately add 950 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.<br>
+. Rotate in the 37°C incubator for 1 h.<br>
+. Make appropriate dilutions in SOC and plate.<br>
+. Incubate at 37°C for 18 h.<br>
 <br>

Team:CU-Boulder/Notebook/Protocols

From 2012.igem.org

Latest revision as of 21:05, 25 June 2012

Protocols