Team:SDU-Denmark/protocols

From 2010.igem.org

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(CP1.3)
 
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<p style="text-align: justify;">
<p style="text-align: justify;">
<br>
<br>
-
How to amplify DNA from bacteria colonies and solutions <br><br>
+
How to amplify DNA from bacterial colonies and solutions <br><br>
''Important remarks'' <br><br>
''Important remarks'' <br><br>
All solutions should be kept at ice until run of PCR <br><br>
All solutions should be kept at ice until run of PCR <br><br>
Line 412: Line 412:
</p>
</p>
-
== Making competent cells of E. coli for transformation ==
+
== Making competent cells of ''E. coli'' for transformation ==
=== CC1.1 ===
=== CC1.1 ===
<p style="text-align: justify;">
<p style="text-align: justify;">
Line 425: Line 425:
''Materials''
''Materials''
<br>
<br>
-
ON culture of TOP10 cells in LB media <br><br>
+
Overnight culture of TOP10 cells in LB media <br><br>
• Ice cold 50mM CaCl2 <br><br>
• Ice cold 50mM CaCl2 <br><br>
• LB media (pre-heated to 37°C)<br><br>
• LB media (pre-heated to 37°C)<br><br>
Line 439: Line 439:
8. Discard the supernatant and resuspend cells in 1.2 ml of icecold CaCl2 (keep the cells on ice!)<br><br>
8. Discard the supernatant and resuspend cells in 1.2 ml of icecold CaCl2 (keep the cells on ice!)<br><br>
9. Leave the cells on ice for 30 min => now the cells are ready for transformation. <br><br>
9. Leave the cells on ice for 30 min => now the cells are ready for transformation. <br><br>
-
=== CC1.2 ===
 
-
<br>
 
-
How to make competent cells for transformation – the iGEM way <br><br>
 
-
''Important remarks''
 
-
<br>
 
-
All of the experiment needs to be carried out in the micro lab <br><br>
 
-
''Materials''
 
-
<br>
 
-
• SOB media (see separate protocol) <br><br>
 
-
• Ice cold CCMB80 buffer <br><br>
 
-
10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) <br><br>
 
-
80 mM CaCl2 .2H2O (11.8g/L)<br><br>
 
-
20 mM MnCl2.4H2O (4.0 g/L)<br><br>
 
-
10 mM MgCl2.6H2O (2.0 g/L)<br><br>
 
-
10% glycerol (100 mL/L)<br><br>
 
-
Adjust pH down to 6.4 with 0.1 M HCl if nessessary. <br><br>
 
-
Adjusting pH down will precipitate manganese dioxide from Mn containing solutions. <br><br>
 
-
Sterile filter and store at 4°C. Slight dark precipitate appears not to affect its function. <br><br>
 
-
• Top10 cells grown on SOB plate <br><br>
 
-
• Glycerol <br><br>
 
-
• SOC (see separate protocol)<br><br>
 
-
''Protocol''
 
-
<br>
 
-
Preparing seed stocks. <br><br>
 
-
1. Streak Top10 cells on an SOB plate and grow for single colonies at 23°C. (room temperature)<br><br>
 
-
2. Pick single colonies into 2 mL of SOB medium and shake overnight at 23°C. <br><br>
 
-
3. Add glycerol to 15% <br><br>
 
-
4. Aliquot 1mL samples to Nunc cryotubes <br><br>
 
-
5. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 min. (this step may not be necessary)<br><br>
 
-
6. Place in -80°C freezer indefinetly <br><br>
 
-
''Preparing competent cells''
 
-
<br>
 
-
1. Inoculate 250 mL of SOB medium with 1 mL vial of seed stock and grow at 20°C to an OD600nm of 0.3. (This takes approximately 16 h.) You can adjust this temperature somewhat to fit your schedule aim for lower, not higher OD if you cannot hit this mark. <br><br>
 
-
2. Cebtrifuge at 3000g at 4°C for 10 min. in a flat bottom centrifuge bottle (flat bottom centrifuge tubes make the fragile cells easier to resuspend pellets by mixing before adding large amounts of buffer). <br><br>
 
-
3. Gently resuspend in 80 mL of ice cold CCMB80 buffer. (sometimes this is less than completely gentle. It still works). <br><br>
 
-
4. Incubate on ice for 20 min. <br><br>
 
-
5. Centrifuge again at 4°C and resuspend in 10 mL of ice cold CCMB80 buffer.<br><br>
 
-
6. Test OD of a mixture of 200 µL SOC and 50 µL of the resuspended cells. <br><br>
 
-
7. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br><br>
 
-
8. Incubate on ice for 20 min. <br><br>
 
-
9. Aliquot to chilled screw top 2 mL vials or 50 µL into chilled microtiter plates. <br><br>
 
-
10. Store at -80°C indefinitely.<br><br>
 
-
11. '''Optional:''' Test competence.<br><br>
 
-
</p>
 
-
=== Measurement of competence ===
 
-
<p style="text-align: justify;">
 
-
<br>
 
-
How to test transformation efficiency of competent cells – the iGEM way <br><br>
 
-
''Materials''
 
-
<br>
 
-
• pUC19 plasmid (Invitrogen)<br><br>
 
-
• SOC medium (see separate protocol)<br><br>
 
-
''Protocol''
 
-
<br>
 
-
1. Transform 50 µL of competent cells with 1 µL of standard pUC19 plasmid. This is at 10 pg/µL or 10-5 µg/µL (This can be done by diluting 1 µL of NEB pUC19 plasmid (1 µg/µL, NEB part number NS3401S) into 100 mL of TE))<br><br>
 
-
2. Keep on ice for 30 min. <br><br>
 
-
3. Heat shock 60 s. at 42°C '''(Very important)''' <br><br>
 
-
4. Add 250 µL SOC medium <br><br>
 
-
5. Incubate at 37°C for 1 h. in 2 mL centrifuge tubes. (these tubes works well with transformation)<br>
 
-
- Transformation with plasmids pSB1AC3 and pSB1AT3, which are chloramphenicol and tetracycline resistant, incubating for 2 h. yields many more colonies. <br><br>
 
-
6. Plate 20 µL on AMP plates and spread. <br><br>
 
-
7. Incabate plates at 37°C over night. <br><br>
 
-
8. Count the colonies (good cells should yield around 100-400 colonies)
 
-
Transformation efficiency is (dilution factor = 15) x colony x 105/µg DNA <br><br>
 
-
We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br>
 
-
</p>
 
== Transformation ==
== Transformation ==
=== TR1.1 ===
=== TR1.1 ===
-
<p style="text-align: justify;">
 
-
<br>
 
-
How to transform compotent cells
 
<br>
<br>
''Important remarks''
''Important remarks''
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<br>
<br>
'''Positive control with your uncut vector''' <br><br>
'''Positive control with your uncut vector''' <br><br>
-
'''Negative control with no DNA''' <br><br>
+
'''Negative control with no inserted DNA''' <br><br>
''Materials''  
''Materials''  
<br>
<br>
-
• Freshly made compotent E. coli cells. <br><br>
+
• Freshly made compotent ''E. coli'' cells. <br><br>
• LA plates with appropriate antibiotics <br><br>
• LA plates with appropriate antibiotics <br><br>
• LB media <br><br>
• LB media <br><br>
Line 530: Line 461:
<br>
<br>
1. Transfer 5 µl DNA (plasmid or ligation mix) to precooled eppendorf tubes. (Use only 1ul if DNA is taken from distribution plates.) <br><br>
1. Transfer 5 µl DNA (plasmid or ligation mix) to precooled eppendorf tubes. (Use only 1ul if DNA is taken from distribution plates.) <br><br>
-
2. Transfer 200 µl of cells with to the tube and mix by pipetting up and down '''(keep the cells on ice at all times)''' <br><br>
+
2. Transfer 200 µl of competent ''E. coli'' cells to the tube and mix by pipetting up and down '''(keep the cells on ice at all times)''' <br><br>
3. Leave on ice for 30 min. <br><br>
3. Leave on ice for 30 min. <br><br>
4. Heat shock for 90 sec. at 42°C in a water bath, do not shake tubes. <br><br>
4. Heat shock for 90 sec. at 42°C in a water bath, do not shake tubes. <br><br>
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10. Resuspend cells and plate out on LA plates with appropriate antibiotics. <br><br>
10. Resuspend cells and plate out on LA plates with appropriate antibiotics. <br><br>
11. Incubate all plates ON at 37°C <br><br>
11. Incubate all plates ON at 37°C <br><br>
-
</p>
+
 
== Restriction digest ==
== Restriction digest ==
=== RD1.1 ===
=== RD1.1 ===
-
<p style="text-align: justify;">
 
<br>
<br>
-
How to digest DNA using fast digest restriction enzymes. <br><br>
+
How to digest DNA using Fermentas fast digest restriction enzymes. <br><br>
''Important remarks''  
''Important remarks''  
<br>
<br>
-
Remember to load a documentation slot next to the marker and take a picture of this for later documentation. <br><br>
+
Remember to load 2-5µL uncut product next to the marker and take a picture of this for later documentation. <br><br>
''Materials''  
''Materials''  
<br>
<br>
Line 558: Line 488:
• 2 µL Fast Digest green buffer <br><br>
• 2 µL Fast Digest green buffer <br><br>
• 5 µL PCR product <br><br>
• 5 µL PCR product <br><br>
-
Multiply protocol if more digested PCR product is needed <br><br>
+
Multiply restriction mixtures if more digested PCR product is needed <br><br>
''Protocols'' <br>
''Protocols'' <br>
-
1. Prepare a purification gel <br><br>
+
1. Cast an agarose gel of suitable percentage for purification of the cut product <br><br>
2. Mix the restriction mixture in en eppendorf tube by pipetting up and down <br><br>
2. Mix the restriction mixture in en eppendorf tube by pipetting up and down <br><br>
3. Leave for 5 min. at 37°C (no shaking!)<br><br>
3. Leave for 5 min. at 37°C (no shaking!)<br><br>
4. Immidiately load the restriction mixture in the gel <br><br>
4. Immidiately load the restriction mixture in the gel <br><br>
-
5. Run the gel and perform a purification step <br><br>
+
5. Run the gel and cut out and purify correct sized bands. <br><br>
</p>
</p>
 +
== Ligation ==
== Ligation ==
-
=== LG1.1 ===
+
 
-
<p style="text-align: justify;">
+
-
<br>
+
-
How to assemble DNA biobricks <br><br>
+
-
''Materials''
+
-
<br>
+
-
Ligation mixture: <br><br>
+
-
For 1 ligation reaction <br><br>
+
-
• 2 µL 10x T4 ligase buffer <br><br>
+
-
• 1 µL T4 ligase (add last!)<br><br>
+
-
• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks <br><br>
+
-
''Protocol''
+
-
<br>
+
-
1. Prepare the ligation mixture and mix by pipetting up and down <br><br>
+
-
2. Leave the mixture over-night at 17°C <br><br>
+
-
3. Test ligation using TAQ-PCR and run test gel afterwards in order to check that the PCR product has the right size <br><br>
+
-
</p>
+
=== LG1.2 ===
=== LG1.2 ===
<p style="text-align: justify;">
<p style="text-align: justify;">
Line 592: Line 507:
Ligation mixture: <br><br>
Ligation mixture: <br><br>
For 1 ligation reaction <br><br>
For 1 ligation reaction <br><br>
-
• 2 µL 10x T4 ligase buffer <br><br>
+
• 2 µL 10x T4 DNA ligase buffer <br><br>
-
• 1 µL T4 ligase (add last!)<br><br>
+
• 1 µL T4 DNA ligase (add last!)<br><br>
• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks <br><br>
• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks <br><br>
-
• Add H2O to reach a total volume of 20mL<br><br>
+
• Add H2O to reach a total volume of 20µL<br><br>
''Protocol''
''Protocol''
<br>
<br>
1. Prepare the ligation mixture and mix by pipetting up and down <br><br>
1. Prepare the ligation mixture and mix by pipetting up and down <br><br>
-
2. Leave the mixture over-night at 17°C <br><br>
+
2. Leave the mixture overnight at 17°C <br><br>
-
3. Test ligation using TAQ-PCR and run test gel afterwards in order to check that the PCR product has the right size <br><br>
+
2a.     If there is no time leave the ligation solution at 22.5&deg;C for 30mins. Then denature the ligase at 65&deg;C for 10min. <br><br>
 +
3.      Use ligation solution for transformations<br>
--[[User:Tipi|Tipi]] 06:48, 20 July 2010 (UTC)
--[[User:Tipi|Tipi]] 06:48, 20 July 2010 (UTC)
</p>
</p>
-
=== LG1.3 ===
 
-
<p style="text-align: justify;">
 
-
''Materials''
 
-
*[[T4 DNA ligase]]
 
-
*10x T4 DNA Ligase Buffer
 
-
*Deionized, sterile H<sub>2</sub>O
 
-
*Purified, linearized vector (likely in H<sub>2</sub>O or EB)
 
-
*Purified, linearized insert (likely in H<sub>2</sub>O or EB)
 
-
''Equipment''
+
== DNA extraction from gel ==
-
Vortex
+
-
 
+
-
''Procedure''
+
-
'''10&mu;L Ligation Mix'''
+
-
''Larger ligation mixes are also commonly used''
+
-
*1.0 &mu;L 10X T4 ligase buffer
+
-
*6:1 molar ratio of insert to vector (~10ng vector)
+
-
*Add (8.5 - vector and insert volume)&mu;l ddH<sub>2</sub>O
+
-
*0.5 &mu;L T4 Ligase
+
-
<br><br>
+
-
''Calculating Insert Amount''
+
-
<math>{\rm Insert\ Mass\ in\ ng} = 6\times\left[\frac{{\rm Insert\ Length\ in\ bp}}{{\rm Vector\ Length\ in\ bp}}\right]\times{\rm Vector\ Mass\ in\ ng}</math>
+
-
 
+
-
'''The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1.  It may be necessary to try several ratios in parallel for best results.'''
+
-
 
+
-
''Method''
+
-
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
+
-
#Add 1 &mu;L ligation buffer to the tube.  <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
+
-
#Add appropriate amount of insert to the tube.
+
-
#Add appropriate amount of vector to the tube.
+
-
#Add 0.5 &mu;L ligase. <br>Vortex ligase before pipetting to ensure that it is well-mixed.  <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add only 0.5 &mu;L, just touch your tip to the surface of the liquid when pipetting.
+
-
#Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
+
-
#Denature the ligase at 65&deg;C for 10min
+
-
#Dialyze for 20 minutes if electroporating
+
-
#Use disks shiny side up
+
-
#Store at -20&deg;C
+
-
 
+
-
''Factors affecting efficiency''
+
-
From [[Tom Ellis]]
+
-
 
+
-
A protocol analysis experiment for a typical DNA ligation (7.2 kb vector + 0.6 kb insert, sticky ends) gave optimal ligation efficiency when 50 ng of vector was ligated overnight at 16&deg;C with a 2:1 insert:vector molar ratio and standard T4 ligase. Ligase was heat inactivated at 65&deg;C for 20 mins before 2 &mu;L (of 20 &mu;L) was used to transform commercial heat-shock competent cells.
+
-
 
+
-
Ligation efficiency was '''marginally decreased''' by
+
-
#Doing a 1 hr ligation at room temperature
+
-
#Using 100 ng vector
+
-
#Using insert:vector molar ratios of 5:1 and 1:1
+
-
 
+
-
Ligation efficiency was '''noticably decreased''' (x100) by
+
-
#Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert)
+
-
#Blunt end ligation
+
-
 
+
-
Ligation efficiency was '''severely decreased''' (x10000) by
+
-
#Using DNA fragments that have been exposed to UV during the gel extraction procedure (''can avoid by blind excision, or by using a black-light or 365nm UV transilluminator instead of the usual 312nm type'')
+
-
#Using the NEB Quick Ligation Kit (''heat inactivation of PEG in the buffer ruins transformation, without heat inactivation the ligation probably would've been fine'')
+
-
 
+
-
For additional troublshooting, check out the NEB FAQ page for T4 ligation: [http://www.neb.com/nebecomm/products/faqproductM0202.asp#339]
+
-
 
+
-
''Notes''
+
-
#Make sure the buffer is completely melted and dissolved.  The white precipitate is BSA according to [http://www.neb.com/nebecomm/tech_reference/dna_rna/tips.asp NEB].  Make sure the buffer still smells strongly like "wet dog" (to check if the DTT is still good).
+
-
#Because ligase buffer contains ATP, which is unstable and degraded by multiple freeze/thaw cycles, you may want to make 10-20ul aliquots from the original tube.  Ligase buffer may be [http://www.neb.com/nebecomm/tech_reference/dna_rna/tips.asp spiked] with additional ATP.
+
-
#If you are having trouble with your ligation, NEB offers FAQ's ([http://www.neb.com/nebecomm/products/faqproductM2200.asp Quick Ligation] [http://www.neb.com/nebecomm/products/faqproductM0202.asp T4 DNA ligase]) and [http://www.neb.com/nebecomm/tech_reference/dna_rna/tips.asp tips] to help.
+
-
#Prior to the ligation, some heat their DNA slightly (maybe ~37&deg;C) to melt any sticky ends which may have annealed improperly at low temperatures.
+
-
#[[Tom Knight]] has read that ligase can inhibit transformation <cite>Michelsen-Anal-1995</cite>.  By heat-inactivating the ligase, this inhibition can be avoided.  However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.
+
-
#Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. <cite>Crowe-NAR-1991</cite>
+
-
#Using [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA Gel Stain] is a safer, non-carcinogenic alternative to ethidium bromide.
+
-
#T4 DNA Ligase is very sensitive to shear, so spinning your ligation mix or vortexing it to mix it can affect your yields.  Instead try mixing with the pipette tip or slowly resuspending the solution.
+
-
 
+
-
''Acknowledgments''
+
-
This protocol is primarily based on [[Endy:DNA ligation using T4 DNA ligase]].
+
-
 
+
-
''References''
+
-
<biblio>
+
-
# Crowe-NAR-1991 pmid=2011503
+
-
# Olivera-PNAS-1967 pmid=5341238
+
-
// DNA ligation by ''Escherichia coli'' DNA ligase
+
-
# Michelsen-Anal-1995 pmid=7778774
+
-
 
+
-
</biblio>
+
-
</p>
+
-
== DNA extraction from gel (fermentas) ==
+
=== DE1.1 ===
=== DE1.1 ===
-
<p style="text-align: justify;">
+
Gel extractions were done according to the
-
<br>
+
[http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0691.pdf protocol] of Fermentas with the exception that we introduced an additional centrifugation step after washing to remove surplus ethanol. <br>
-
How to extract and purify DNA from gel <br><br>
+
=== DE1.3 ===
-
''Important remarks''
+
Gel extractions were done according to the [http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/7D3C39CAF8206AD1C1257628001D5012/$file/28951562AA.pdf protocol] of GE Healthcare with the exception that we introduced an additional centrifugation step after washing to remove surplus ethanol.
-
<br>
+
-
All steps should be carried out at room temperature. <br><br>
+
-
All centrifugations should be carried out in a table-top microcentrifuge at >12000x g <br><br>
+
-
''Materials''
+
-
<br>
+
-
• Binding buffer <br><br>
+
-
• Wash buffer (diluted with ethanol)<br><br>
+
-
• Elution buffer <br><br>
+
-
''Protocol''
+
-
<br>
+
-
1. Weigh a 1.5 µL tube <br><br>
+
-
2. Excise gel slice containing the DNA fragment using a clean scalpel (cut as close to the DNA as possible)<br><br>
+
-
3. Place the gel slice into the pre-weighed tube and record the weight of the gel slice <br><br>
+
-
4. Add 1:1 volume of binding buffer to the gel slice (e.g. add 100 µL of binding buffer for every 100 mg of agarose gel)<br><br>
+
-
5. Incubate the gel mixture at 50-60°C for 10 min. or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes. ''The color of the solution should be yellow. If the color of the solution is orange or violet add 10 µL 3M sodium acetate , pH 5.2 and mix. The color will then turn yellow.'' <br><br>
+
-
6. Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube. <br><br>
+
-
7. Add 700 µL of Wash buffer to the column. Centrifuge for 1 min. Discard flow-through and place the column back into the collection tube. <br><br>
+
-
8. Centrifuge the empty column for an additional 1 min. to completely remove residual Wash buffer ''This step is essential to avoid residual ethanol in the purified DNA solution'' <br><br>
+
-
9. Transfer the column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane. Centrifuge for 1 min. <br><br>
+
-
10. Discard the column and store the purified DNA at -20°C. <br><br>
+
-
</p>
 
-
=== DE1.2 ===
 
-
<p style="text-align: justify;">
 
<br>
<br>
-
How to extract and purify DNA from gel <br><br>
 
-
''Important remarks''
 
-
<br>
 
-
All steps should be carried out at room temperature. <br><br>
 
-
All centrifugations should be carried out in a table-top microcentrifuge at >12000x g <br><br>
 
-
''Materials''
 
-
<br>
 
-
• Binding buffer <br><br>
 
-
• Wash buffer (diluted with ethanol)<br><br>
 
-
• Elution buffer <br><br>
 
-
''Protocol''
 
-
<br>
 
-
1. Weigh a 1.5 µL tube <br><br>
 
-
2. Excise gel slice containing the DNA fragment using a clean scalpel (cut as close to the DNA as possible)<br><br>
 
-
3. Place the gel slice into the pre-weighed tube and record the weight of the gel slice <br><br>
 
-
4. Add 1:1 volume of binding buffer to the gel slice (e.g. add 100 µL of binding buffer for every 100 mg of agarose gel)<br><br>
 
-
5. Incubate the gel mixture at 50-60°C for 10 min. or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes. ''The color of the solution should be yellow. If the color of the solution is orange or violet add 10 µL 3M sodium acetate , pH 5.2 and mix. The color will then turn yellow.'' <br><br>
 
-
6. Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube. <br><br>
 
-
7. Add 700 µL of Wash buffer to the column. Centrifuge for 1 min. Discard flow-through and place the column back into the collection tube. <br><br>
 
-
8. Centrifuge the empty column for an additional 1 min. to completely remove residual Wash buffer ''This step is essential to avoid residual ethanol in the purified DNA solution'' <br><br>
 
-
9. Transfer the column into a clean 1.5 mL microcentrifuge tube. Add 20 µL of H2O to the center of the column membrane. Centrifuge for 1 min. <br><br>
 
-
10. Discard the column and store the purified DNA at -20°C. <br><br>
 
-
--[[User:Tipi|Tipi]] 06:45, 20 July 2010 (UTC)
 
-
</p>
 
-
=== DE1.3 ===
 
-
<p style="text-align: justify;">
 
-
<br>
 
-
'''Protocol for purification of DNA from TAE and TBE agarose gel bands'''<br>
 
-
[http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=39955 Kit from GFX].<br><br>
 
-
'''''Sampla capture'''''<br>
 
-
1. Weigh a DNase-free 1.5 ml microcentrifuge tube.<br><br>
 
-
2. Exice band of interest from the gel and place in microcentrifuge tube.<br><br>
 
-
3. Weigh microcentrifuge tube plus agarose gel band.<br><br>
 
-
4. Calculate weight of agarose gel slice.<br><br>
 
-
5. Add 10 ul Capture buffer type 3 for each 10 mg agarose slive ('''add at least 300 ul!''')<br><br>
 
-
6. Mix by inversion. <br><br>
 
-
7. Place at 60 degrees celcius until agarose is completely dissolved.<br><br>
 
-
'''''Sample binding'''''<br>
 
-
1. Add up to 600 ul Capture buffer-sample mix to assembled GFX MicroSpin columns and Collection tube.<br><br>
 
-
2. Leave at room temperature for 1 minute.<br><br>
 
-
3. Centrifuge for 30 sec. at 16,000g.<br><br>
 
-
4. Discard the flow-through in the Collection tube and place the MicroSpin column in the collection tube again.<br><br>
 
-
5. Repeat sample binding step until all sample is loaded onto the MicroSpin column.<br><br>
 
-
 
-
''''' Wash and Dry '''''<br>
 
-
1. Add 500 ul Wash buffer type 1.<br><br>
 
-
2. Centrifuge for 30 sec. at 16,000g.<br><br>
 
-
3. Discard flow-through and keep Collection tube a above.<br><br>
 
-
4. Centrifuge again for 30 sec. at 16,000g. <br>'''More flow-through will appear in the collection tube. It is important to centrifuge this second time to get the sample completely dry. This step is not included in the original protocol.''' <br><br>
 
-
5. Discard Collection tube and trensfer MicroSpin column to a clean 1.5 ml DNase-free microcentrifuge tube.<br><br>
 
-
 
-
''''' Elution '''''<br>
 
-
1. Add 10 - 50 ul Elution buffer type 4 or 6. ('''10 ul is fine for small volumes''')<br><br>
 
-
2. Leave at room temperature for 60 sec.<br><br>
 
-
3. Centrifuge for 1 min. at 16,000g.<br><br>
 
-
4. Retain flow-through and discard MicroSpin Column.<br><br>
 
-
5. Store purified sample DNA at -20 degrees or proceed to cutting DNA og ligation.<br><br>
 
-
</p>
 
== Genomic DNA purification ==
== Genomic DNA purification ==
=== GP1.1 ===
=== GP1.1 ===
Line 782: Line 539:
All steps should be carried out at room temperature. <br><br>
All steps should be carried out at room temperature. <br><br>
Be sure to mix thoroughly when adding the solutions. <br><br>
Be sure to mix thoroughly when adding the solutions. <br><br>
-
Addition and removal of chloroform should be carried out in fume hood. <br><br>
+
Addition and removal of chloroform should be carried out in a fume hood. <br><br>
''Materials''
''Materials''
<br>
<br>
Line 793: Line 550:
''Protocol''
''Protocol''
<br>
<br>
-
1. Mix 200 µL of sample (ON culture) with 400 µL of Lysis solution and incubate at 65°C for 5 min.''If a frozen sample is used lysis solution should be added before thawing and incubated at 65°Cfor 10 min. with occasional inverting the tube.'' <br><br>
+
1. Mix 200 µL of sample (overnight culture) with 400 µL Lysis solution and incubate at 65°C for 5 min.''If a frozen sample is used lysis solution should be added before thawing and incubated at 65°Cfor 10 min. with occasional inverting the tube.'' <br><br>
2. Immediately add 600 µL of chloroform, gently emulsify by inversion (3-5 times) and centrifuge the sample at 10.000 rpm for 2 min. <br><br>
2. Immediately add 600 µL of chloroform, gently emulsify by inversion (3-5 times) and centrifuge the sample at 10.000 rpm for 2 min. <br><br>
3. Prepare precipitation solution. <br><br>
3. Prepare precipitation solution. <br><br>
Line 802: Line 559:
8. Store DNA at -20°C <br><br>
8. Store DNA at -20°C <br><br>
</p>
</p>
-
== Plasmid miniprep kit (Fermentas) ==
+
 
 +
== Plasmid miniprep kit ==
=== MP1.1 ===
=== MP1.1 ===
-
<p style="text-align: justify;">
+
 
-
<br>
+
Plasmids are isolated from cultures by transferring 5 mL overnight culture to a 15 mL falcon tube and spinning down at 4000g for 15 min and removing supernatant. <br><br>
-
How to isolate plasmids from cultures <br><br>
+
 
-
''Important remarks''
+
Then the GeneJet Plasmid miniprep kit  from Fermantas was used according to manufacturers [http://fermentas.com/templates/files/tiny_mce/media_pdf/broch_genejet_P19.pdf recommendations]. <br>
-
<br>
+
-
All steps should be carried out at room temperature. <br><br>
+
-
Step 1 and 2 must be carried out in the micro lab. <br><br>
+
-
''Materials''
+
-
<br>
+
-
• Resuspension solution (with RNase A) <br><br>
+
-
• Lysis solution <br><br>
+
-
• Neutralization solution <br><br>
+
-
• Wash solution (diluted with ethanol)<br><br>
+
-
• Elution solution <br><br>
+
-
''Protocol''
+
-
<br>
+
-
1. Resuspend pelleted cells in 250 µL Resuspension solution. Resuspend completely by vortexing. Transfer the cell suspension to microcentrifuge tubes. <br><br>
+
-
2. Add 250 µL Lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution is viscous and slighty clear. (Do not vortex!)<br><br>
+
-
3. Add 350 µL Neutralization buffer and mix immediately and thoroughly by inverting the tube 4-6 times. (It is important to mix gently to avoid localized precipitation)<br><br>
+
-
4. Centrifuge for 5 min. to pellet cell debris and chromosomal DNA. <br><br>
+
-
5. Transfer supernatant to the supplied GeneJet spin column, without disturbing or transferring the white precipitate. <br><br>
+
-
6. Cenntrifuge for 1 min. Discard flow-through and place column back into the same collection tube. <br><br>
+
-
7. Add 500 µL Wash solution to the column. Centrifuge for 30-60 s. and discard the flow-through. Place column back into the same tube. <br><br>
+
-
8. Repeat step 7. <br><br>
+
-
9. Discard the flow-through and centrifuge for an additional 1 min. to remove residual wash solution. (This step is essential to avoid residual ethanol in plasmid preps) <br><br>
+
-
10. Transfer the column into a fresh 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane to elute the plasmid DNA (do not touch the membrane with the pipette tip!). Incubate for 2 min. at room temperature and centrifuge for 2 min. <br>
+
-
Optional: repeat elution step to increase the overall yield by 10-20%. <br><br>
+
-
11. Discard the column and store the purified plasmid DNA at -20°C. <br><br>
+
-
</p>
+
=== MP1.2 ===
=== MP1.2 ===
-
<p style="text-align: justify;">
+
 
-
<br>
+
Plasmids are isolated from cultures by transferring 10 mL overnight culture to a 15 mL falcon tube and spinning down at 4000g for 15 min and removing supernatant. <br><br>
-
How to isolate plasmids from cultures <br><br>
+
 
-
''Important remarks''
+
Then the GeneJet Plasmid miniprep kit  from Fermantas was used according to manufacturers [http://fermentas.com/templates/files/tiny_mce/media_pdf/broch_genejet_P19.pdf recommendations]. <br>
-
<br>
+
 
-
All steps should be carried out at room temperature. <br><br>
+
-
Step 1 and 2 must be carried out in the micro lab. <br><br>
+
-
''Materials''
+
-
<br>
+
-
• Resuspension solution (with RNase A) <br><br>
+
-
• Lysis solution <br><br>
+
-
• Neutralization solution <br><br>
+
-
• Wash solution (diluted with ethanol)<br><br>
+
-
• Elution solution <br><br>
+
-
''Protocol''
+
-
<br>
+
-
1.      Transfer 10 mL ON-culture to a 15 mL falcon tube and spin down at 4000g for 15 min. <br><br>
+
-
2. Resuspend pelleted cells in 500 µL Resuspension solution. Resuspend completely by vortexing. Divide the cell suspension in 2x250ul and transfer to eppendorf tubes. From now on proceed with the two tubes in parallel. <br><br>
+
-
3. Add 250 µL Lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution is viscous and slighty clear. (Do not vortex!)<br><br>
+
-
4. Add 350 µL Neutralization buffer and mix immediately and thoroughly by inverting the tube 4-6 times. (It is important to mix gently to avoid localized precipitation)<br><br>
+
-
5. Centrifuge for 5 min. to pellet cell debris and chromosomal DNA. <br><br>
+
-
6. Transfer supernatant to the supplied GeneJet spin column, without disturbing or transferring the white precipitate. <br><br>
+
-
7. Cenntrifuge for 1 min. Discard flow-through and place column back into the same collection tube. <br><br>
+
-
8. Add 500 µL Wash solution to the column. Centrifuge for 30-60 s. and discard the flow-through. Place column back into the same tube. <br><br>
+
-
9. Repeat step 8. <br><br>
+
-
10. Discard the flow-through and centrifuge for an additional 1 min. to remove residual wash solution. (This step is essential to avoid residual ethanol in plasmid preps) <br><br>
+
-
11. Transfer the column into a fresh 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane to elute the plasmid DNA (do not touch the membrane with the pipette tip!). Incubate for 2 min. at room temperature and centrifuge for 2 min. <br>
+
-
Optional: repeat elution step to increase the overall yield by 10-20%. <br><br>
+
-
12. Discard the column and store the purified plasmid DNA at -20°C. <br><br>
+
-
</p>
+
=== MP1.3 ===
=== MP1.3 ===
-
<p style="text-align: justify;">
+
 
 +
Plasmids are isolated from exponential growing cultures by: <br>
 +
1.      Transferring 2.5mL overnight culture to 15mL preheated LB medium and growing cells for an additionally 2 hours.<br><br>
 +
2. Transfer all 17.5mL new culture to a 50mL falcon tube and spin down at 4000g for 15 min. <br><br>
 +
 
 +
Proceed with the the GeneJet Plasmid miniprep kit from Fermantas was used according to manufacturers [http://fermentas.com/templates/files/tiny_mce/media_pdf/broch_genejet_P19.pdf recommendations]. <br>
<br>
<br>
-
How to isolate plasmids from cultures <br><br>
+
 
-
''Important remarks''
+
== Preparation of Agarose gelmix for gel electrophoresis ==
-
<br>
+
-
All steps should be carried out at room temperature. <br><br>
+
-
Step 1 and 2 must be carried out in the micro lab. <br><br>
+
-
''Materials''
+
-
<br>
+
-
• Resuspension solution (with RNase A) <br><br>
+
-
• Lysis solution <br><br>
+
-
• Neutralization solution <br><br>
+
-
• Wash solution (diluted with ethanol)<br><br>
+
-
• Elution solution <br><br>
+
-
''Protocol''
+
-
<br>
+
-
1.      Cells from ON culture is reboosted by transfering 2.5mL ON culture to 15mL LB preheated LB medium.Cells are then grown for    additionally 2 hours.<br><br>
+
-
2. Transfer all 17.5mL new culture to a 50mL falcon tube10 mL ON-culture and spin down at 4000g for 15 min. <br><br>
+
-
3. Resuspend pelleted cells in 250 µL Resuspension solution. Resuspend completely by vortexing.<br><br>
+
-
4. Add 250 µL Lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution is viscous and slighty clear. (Do not vortex!)<br><br>
+
-
5. Add 350 µL Neutralization buffer and mix immediately and thoroughly by inverting the tube 4-6 times. (It is important to mix gently to avoid localized precipitation)<br><br>
+
-
6. Centrifuge for 5 min. to pellet cell debris and chromosomal DNA. <br><br>
+
-
7. Transfer supernatant to the supplied GeneJet spin column, without disturbing or transferring the white precipitate. <br><br>
+
-
8. Cenntrifuge for 1 min. Discard flow-through and place column back into the same collection tube. <br><br>
+
-
9. Add 500 µL Wash solution to the column. Centrifuge for 30-60 s. and discard the flow-through. Place column back into the same tube. <br><br>
+
-
10. Repeat step 8. <br><br>
+
-
11. Discard the flow-through and centrifuge for an additional 1 min. to remove residual wash solution. (This step is essential to avoid residual ethanol in plasmid preps) <br><br>
+
-
12. Transfer the column into a fresh 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane to elute the plasmid DNA (do not touch the membrane with the pipette tip!). Incubate for 2 min. at room temperature and centrifuge for 2 min. <br>
+
-
Optional: repeat elution step to increase the overall yield by 10-20%. <br><br>
+
-
13. Discard the column and store the purified plasmid DNA at -20°C. <br><br>
+
-
</p>
+
-
== Preparation of Agarose for gel electrophoresis ==
+
=== AG1.1 ===
=== AG1.1 ===
-
<p style="text-align: justify;">
+
 
-
<br>
+
-
How to prepare Agarose for gel electrophoresis.<br><br>
+
''Important remarks''
''Important remarks''
<br>
<br>
Agarose concentration is dependent on the size of the DNA fragment that needs to be seperated (see the door of the incubator in the gel room) <br><br>
Agarose concentration is dependent on the size of the DNA fragment that needs to be seperated (see the door of the incubator in the gel room) <br><br>
-
Addition of EtBr is carried out in fume hood. <br><br>
+
Addition of EtBr is carried out in a fume hood. <br><br>
''Materials''
''Materials''
<br>
<br>
Line 915: Line 598:
2. The mixture is heated for 5 min. at max temperature in micro-wave. ''Remember to note name, date and –EtBr on the flask.'' <br><br>
2. The mixture is heated for 5 min. at max temperature in micro-wave. ''Remember to note name, date and –EtBr on the flask.'' <br><br>
3. Place flask in the incubator for 20 min or at room temperature until cooled to 60°C. <br><br>
3. Place flask in the incubator for 20 min or at room temperature until cooled to 60°C. <br><br>
-
4. Add 5 droplets of EtBr. <br><br>
+
4. Add 5 droplets of EtBr and mix. <br><br>
-
5. Cast gel and leave for 20 min until the gel is set. ''Remaining agarose solution is placed in incubater for later use.'' <br><br>
+
5. Cast gel and leave for 20 min until the gel is set. ''Remaining agarose solution is placed in incubator for later use.'' <br><br>
-
6. Load gel and run gel. ''Load only 5 µL of DNA marker'' <br><br>
+
6. Load and run gel. ''Load 5 µL of DNA marker'' <br><br>
-
</p>
+
 
-
== Preparation of SOB and SOC media ==
+
-
<br>
+
-
How to prepare SOB and SOC media for transformation. <br><br>
+
-
=== SOB medium ===
+
-
<p style="text-align: justify;">
+
-
<br>
+
-
Used in growing bacteria for preparing chemically compotent cells. <br><br>
+
-
''Materials''
+
-
<br>
+
-
For 1 L:<br>
+
-
• 20 g tryptone <br><br>
+
-
• 5 g yeast extract <br><br>
+
-
• 0.5g NaCl <br><br>
+
-
• dH2O to 1 L <br><br>
+
-
• KCl (is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O)<br><br>
+
-
• 2M MgCl2 (is made by dissolving 19g MgCl2 in 90 mL dH2O =>adjust to obtain a volume of 100 mL using dH2O => autoclavate)<br><br>
+
-
''Protocol''
+
-
<br>
+
-
1. Add tryptone, yeast extract and NaCl to 950 mL of dH2O and shake until solute has dissolved. <br><br>
+
-
2. Add 10 mL of 250 mM solution of KCl <br><br>
+
-
3. Adjust volume to 1 L using dH2O <br><br>
+
-
4. Autoclavate for 20 min. <br><br>
+
-
5. Just before use add 5 mL of sterile solution of 2M MgCl2 <br><br>
+
-
</p>
+
-
=== SOC medium ===
+
-
<p style="text-align: justify;">
+
-
<br>
+
-
''Materials''
+
-
<br>
+
-
• SOB medium. <br><br>
+
-
• 1M glucose (is made by dissolving 18g of glucose in 90 mL of dH2O => adjust to obtain a volume of 100 mL using dH2O)<br><br>
+
-
''Protocol''
+
-
<br>
+
-
1. Cool SOB medium to 60°C <br><br>
+
-
2. Add 20mL of 1M glucose. <br><br>
+
-
</p>
+
== Extraction af Carotenoids and polyene chromophores ==
== Extraction af Carotenoids and polyene chromophores ==
=== EX1.1 ===
=== EX1.1 ===
<p style="text-align: justify;">
<p style="text-align: justify;">
-
1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 oC for 20 hours<br>  
+
1. Incubate ''E. Coli'' in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours<br> <br>
-
2. Harvest cells using centrifugation at 4000 G for 15 min<br>
+
2. Harvest cells by centrifugation at 4000g for 15 min<br><br>
-
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br>  
+
3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
-
4. Centrifuge the samples at 16000 G for 2 min and collect 2 mL of the supernatant<br>
+
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
-
5. Measure absorbance using UV-Vis spectrophotometer at 450 nm, was preformed on a …. From MEMPHYS<br>
+
5. Measure absorbance using UV-Vis spectrophotometer at 450 nm  
 +
<br><br>
</p>
</p>
 +
=== EX1.2 ===
=== EX1.2 ===
<p style="text-align: justify;">
<p style="text-align: justify;">
-
1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 oC for 20 hours<br>  
+
1. Incubate ''E. Coli'' in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours<br> <br>
-
2. Harvest cells using centrifugation at 4000 G for 15 min<br>
+
2. Harvest cells by centrifugation at 4000g for 15 min<br><br>
-
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br>  
+
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
-
4. Centrifuge the samples at 16000 G for 2 min and collect 2 mL of the supernatant<br>
+
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
-
5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, using a C18… column with 100% Methanol as the A Buffer and 60% Methanol, 40% acetone as the B buffer <br><br>
+
5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, For this particular purpose, we use a Poroshell 120 EC-C18 (4,6 x 150 mm 2,7 micron)column, and the eluents used are as follows: <br>
 +
A-buffer: 100% methanol with 0,1% trifluoroacetic acid. <br>
 +
B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid. <br>
 +
Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer. <br>
 +
Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. Usage of the HPLC and instruction on how to use it was kindly provided by [http://www.sdu.dk/Om_SDU/Institutter_centre/C_FLinT FLINT]<br>
 +
<br>
</p>
</p>
Line 980: Line 634:
• Diluted LB media<br>
• Diluted LB media<br>
• Difco Agar<br>
• Difco Agar<br>
-
1mM retinal<br><br>
+
1µM retinal (final concentration)<br><br>
''Swimming motility plates''<br>
''Swimming motility plates''<br>
-
1. LB media is mixed with 0.3% difco agar and is autoclavated<br>
+
1. LB media is mixed with 0.3% difco agar and autoclavated<br><br>
-
2. The appropriate antibiotic and 1uM retinal is added to the autoclaved media (NB: for the media used for the control plates, no antibiotic or retinal is added)<br>
+
2. The appropriate antibiotic and 1µM retinal (final concentration) is added to the autoclaved media (NB: for the media used for the control plates, no antibiotic or retinal is added)<br><br>
-
3. Plates are cast and incubated ON at room temperature<br>
+
3. Plates are cast and incubated overnight at room temperature<br><br>
-
4. 15 minutes prior to the experiment the plates are incubated at 37°C.<br><br>
+
4. 15 minutes prior to the experiment the plates are dried at 37°C.<br><br>
''Sample preparation''<br>
''Sample preparation''<br>
-
1. Colony is inoculated in 5mL LB media containing the appropriate antibiotic. The culture is grown ON at 37°C and 180rpm.<br>
+
1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony. The culture is grown overnight at 37°C and 180rpm.<br>
-
2. ON culture is diluted in 5mL LB media containing the appropriate antibiotic to reach an OD550 of 0.02 and are incubated at 37°C and 180rpm until it reaches an OD550 of 0.5.<br>  
+
2. The overnight culture is diluted in 5mL LB media containing the appropriate antibiotic to reach an OD550 of 0.02 and are incubated at 37°C and 180rpm until it reaches an OD550 of 0.5.<br>  
-
3. 1mM retinal is added to the culture (NB: no retinal is added to the cultures containing the control cells)<br>
+
3. 1µM retinal (final cnocentration) is added to the culture (NB: no retinal is added to the cultures containing the control cells)<br>
-
4. The tubes containing the cultures are wrapped in tin foil and are subsequently grown for 2 hours at 37°C and 180rpm.<br><br>
+
4. The tubes containing the cultures are wrapped in tin foil (creates darkness) and are subsequently grown for 2 hours at 37°C and 180rpm.<br><br>
''Motility assay''<br>  
''Motility assay''<br>  
-
1. 2x2.5uL of culture is placed on each plate, and the plates are placed in a specially engineered lightbox, so that ½ of each plate is illuminated with blue light and the other ½ is kept in dark. 1mL of each culture is used for microscopy.<br>
+
1. 2x2.5µL of culture is placed on each plate, and the plates are placed in a specially engineered lightbox, so that ½ of each plate is illuminated with blue light and the other ½ is kept in dark. <br>
2. The plates are incubated at 37°C for 24 hours.<br>
2. The plates are incubated at 37°C for 24 hours.<br>
3. Pictures are taken<br><br>
3. Pictures are taken<br><br>
''Microscopy''<br>
''Microscopy''<br>
-
1. 5uL cell culture is used for the microscopy<br>
+
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
-
2. To avoid laminar flow, the microscopy slide is sealed with nail polish.<br>
+
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
3. Samples are examined under the microscope.<br><br>
3. Samples are examined under the microscope.<br><br>
 +
 +
=== PS1.2 ===
 +
A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br>
 +
This protocol is designed based on preceeding pilot studies<br>
 +
 +
''Materials:''<br>
 +
• LB media<br>
 +
• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 1]]<br>
 +
• 1mM retinal<br>
 +
''Protocol:''
 +
1. A colony is inoculated in 5mL LB media with appropriate antibiotic.<br>
 +
2. The culture is incubated for 12h. at 22° and 160rpm<br>
 +
3. The cultures containing the unmodified bacteria (controls) is then diluted 1:10 in motility buffer, at which point these are ready for microscopy <br>
 +
4. The culture containing the modified bacteria is added 1uM retinal (final concentration), and is incubated for an additional 2h in darkness at 22°C and 160rpm.<br>
 +
5. The culture is then diluted 1:10 in motility buffer and are ready for microscopy.<br>
 +
 +
''Microscopy''<br>
 +
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
 +
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
 +
3. Samples are examined under the microscope.<br><br>
 +
 +
 +
 +
 +
 +
 +
</p>
</p>
 +
==Growth rate assay==
==Growth rate assay==
===GA1.1===
===GA1.1===
Line 1,011: Line 693:
''Protocol''<br>
''Protocol''<br>
-
1. A colony is inoculated in 5mL LB media containing the appropriate antibiotic.<br>
+
1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony .<br>
2. The culture is incubated over night at 37°C and 180rpm.<br>
2. The culture is incubated over night at 37°C and 180rpm.<br>
-
3. The optical density at 550nm (OD550) of the ON culture is measured and the culture is diluted in 25uL fresh LB media containing the appropriate antibiotic to reach an OD550 of 0.02. The culture is incubated for 24 hours at 37°C and 180rpm.<br>
+
3. The optical density at 550nm (OD550) of the overnight culture is measured and the culture is diluted in 15mL fresh LB media containing the appropriate antibiotic to reach an OD550 of 0.02. The culture is then incubated at 37°C and 180rpm.<br>
4. OD550 of the colony is measured every hour for the first 12 hours, and after 24 hours.<br><br>  
4. OD550 of the colony is measured every hour for the first 12 hours, and after 24 hours.<br><br>  
</p>
</p>
-
==Flagella staining==
+
 
-
===1.1===  
+
== Flagella staining ==
 +
<br>
 +
The following protocols are based on knowledge recived from [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 2]]<br>
 +
===FS1.1===  
<p style="text-align: justify;">
<p style="text-align: justify;">
-
Day 1: The bacteria were grown in 5 ml-LB media ON. The solutions used for staining were prepared.
+
Day 1: The bacteria were grown in 5 ml-LB media overnight. The solutions used for staining were prepared.
<br><br>
<br><br>
Line 1,027: Line 712:
The following components were added in the listed order:<br>
The following components were added in the listed order:<br>
-
• 5 g of tannic acid was dissolved in 9.65 ml distilled water.<br>
+
• 5 g of tannic acid dissolved in 9.65 ml distilled water.<br>
• 150 µl  9% FeCl3<br>
• 150 µl  9% FeCl3<br>
Line 1,039: Line 724:
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.  
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.  
<br><br>
<br><br>
-
Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately OD550 1.  
+
Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately an OD550 of 1.  
<br><br>
<br><br>
Preliminary bacteria work:<br>
Preliminary bacteria work:<br>
Line 1,065: Line 750:
</p>
</p>
-
===1.2===
+
===FS1.2===
<p style="text-align: justify;">
<p style="text-align: justify;">
-
Day 1: Bacteria were platede on agar plates and incubated at 37 degrees ON.  The staining solutions were prepared.<br><br>
+
Day 1: Bacteria were platede on agar plates and incubated at 37 degrees celcius overnight.  The staining solutions were prepared.<br><br>
Solution I: <br>
Solution I: <br>
Line 1,089: Line 774:
   
   
<br>
<br>
-
Day 2: A bacteria colony was dissolved in LB-media.  
+
Day 2: A bacterial colony was dissolved in LB-media.  
<br><br>
<br><br>
Line 1,114: Line 799:
Materials
Materials
<br><br>
<br><br>
-
*         LB media
+
        LB media <br>
-
*         LA plates
+
        LA plates <br>
-
*         LA plates with appropriate antibiotic
+
        LA plates with appropriate antibiotic<br>
-
*         0.9% NaCl
+
        0.9% NaCl<br>
<br>
<br>
   
   
-
Protocol
+
'''Protocol'''
<br><br>
<br><br>
-
1.      A bacteria colony is inoculated in 5mL LB media with appropriate antibiotic.
+
1.      5mL LB media with appropriate antibiotic is inoculated with a bacterial colony.
<br>
<br>
-
2.      Culture is incubated over night at 30°C and 180rpm
+
2.      The culture is incubated over night at 30°C and 180rpm
<br>
<br>
-
3.      100uL of the culture is serial diluted in 900uL 0.9% NaCl to reach a dilution of 107.
+
3.      100µL of the culture is serially diluted in 900µL 0.9% NaCl until a dilution of 10<sup>7</sup> is reached.
<br>
<br>
-
4.      100uL of the 105, 106 and 107 dilution is spreaded on LA plates, and LA plates with the appropriate antibiotic, respectively.
+
4.      100µL of the 10<sup>5</sup>, 10<sup>6</sup> and 10<sup>7</sup> dilutions are spread onto LA plates, and LA plates with the appropriate antibiotic, respectively.
<br>
<br>
5.      Plates are incubated at 37°C for 16-24 hours. The following day the colonies formed on the plates are counted and cfu are determined.
5.      Plates are incubated at 37°C for 16-24 hours. The following day the colonies formed on the plates are counted and cfu are determined.
<br>
<br>
-
6.      500uL of the 105 dilution is added to 4.5mL of fresh LB media without any antibiotics.
+
6.      500µL of the 10<sup>5</sup> dilution is added to 4.5mL of fresh LB media without any antibiotics.
<br>
<br>
7.      The new culture is incubated over night at 30°C and 180rpm
7.      The new culture is incubated over night at 30°C and 180rpm
<br>
<br>
-
8.      The experiment is carried out for 5 days. (NB: antibiotic is only added to the first culture. The remaining days the bacteria are grown in cultures without any antibiotics)
+
8.      The experiment is carried out for 5 days.  
 +
<br>(NB: antibiotic is only added to the first culture. The remaining days the bacteria are grown in cultures without any antibiotics)
<br><br>
<br><br>
 +
==Scanning Electron Microscope ==
==Scanning Electron Microscope ==
===SEM 1.1===
===SEM 1.1===
<br>
<br>
-
Day 1: The bacteria was cultivated ON in 5 ml LB-media at 37 degrees. <br><br>  
+
Day 1: The bacteria was cultivated overnight in 5 ml LB-media at 37 degrees celcius. <br><br>  
-
Day 2: The ON culture was centrifuged  15 min at 4000prm. Afterwards the pellet was resuspended in  distilled water. We aimed to get approximately 106 bacteria in 10 µl solution which was plated on double adhesive tape at the top of the grid. The solution was allowed to air dry and the remaining fluid appeared when sample were exposed to the vacuum in the electron microscope.  <br><br>
+
Day 2: The overnight culture was centrifuged  15 min at 4000prm. Afterwards the pellet was resuspended in  distilled water. We aimed to get approximately 10<sup>6</sup> bacteria in 10 µl solution which was plated on double adhesive tape at the top of the grid. The solution was allowed to air dry and the remaining fluid disappeared as samples were exposed to the vacuum in the electron microscope.  <br><br>
-
The sample was examined with different electron intensity and magnification. We found that the best picture was taken with a electron intensity of 10 kv.
+
The sample was examined with different electron intensity and magnification. We found that the best picture was taken with a electron intensity of 10 kV  and a magnitude on 10kx.<br><br>
 +
== Charactarization of K389016 (VirA/G reporter device mRFP) ==
 +
===CK1.1===
 +
''Materials:''<br>
 +
•    20mM acetosyringone (39.3 acetosyringone dissolved in 1mL DMSO and 9mL ddH2O)<br>
 +
•    LB media<br>
 +
•    35µg/mL chloramphenicol<br>
 +
''Protocol:''<br>
 +
1.    A colony is inoculated in 5mL LB media with 35µg/mL chloramphenicol and incubated over night at 37°C and 180rpm.<br>
 +
2.    Parallel cultivations of 25mL LB media, 35µg/mL chloramphenicol and acetosyringone concentrations of 0uM (control), 100uM, 200uM and 400uM respectively.<br>
 +
3.    The cultures were incubated in a waterbath of 37°C and 180rpm<br>
 +
4.    The optical density at 550nm (OD550) was measured every two hours and samples were freezed at -80°C and used for fluorescence measurements (excitation at 584nm, emmitation at 607nm)<br><br>
 +
== References ==
 +
[1]Khan S, Amoyaw K, Spudich JL, Reid GP, Trentham DR,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1260487/pdf/biophysj00100-0082.pdf Bacterial chemoreceptor signaling probed by flash photorelease of a caged serine], Biophys J. 1992 Apr;62(1)<br>
 +
[2]Harshey RM, Matsuyama T,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44660/pdf/pnas01140-0333.pdf Dimorphic transition in Escherichia coli and Salmonella typhimurium: Surface-induced differentiation into hyperflagellate swarmer cells],Proc Natl Acad Sci U S A. 1994 August 30; 91(18): 8631–8635<br>
</p>
</p>
</div>
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<div id="rightcolumn">
<div id="rightcolumn">
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Almost like cake recipes... although cake is infinitely more delicious than bacteria.
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Almost like cake recipes... although cake is infinitely more delicious than bacteria. <br><br>
 +
Get a template for calculating your ligation concentrations <br><html><a href="https://static.igem.org/mediawiki/2010/4/40/Team_SDUTemplate_for_ligation.ZIP" style="color: #50CC38; text-decoratio: bold; font-size: 24px;">HERE</a></html>
<br>
<br>
<br>
<br>
__TOC__
__TOC__
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<br>
 +
<div id="bonus">
 +
<br>
 +
<br>
 +
<br>
 +
[https://2010.igem.org/Team:SDU-Denmark/project/activities/commoninterest/diller 8===D]
 +
</div>
</div>
</div>

Latest revision as of 03:58, 28 October 2010