Glasgow/Wetlab/Protocols

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(Difference between revisions)
(→Minipreps)
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== Primer Design ==
== Primer Design ==
-
== Minipreps ==
+
== Qiagen Minipreps ==
 +
 
 +
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of ''E. coli'' in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.
 +
 
 +
=== Procedure ===
 +
 
 +
#Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube.
 +
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
 +
#Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
 +
#Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times.
 +
To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
 +
#Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
 +
#Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
 +
#Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results.
 +
#(Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.
 +
#Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Spinning for 60 seconds produces good results.
 +
#Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
 +
IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.
 +
#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 each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. If you are concerned about the concentration of the DNA, you can alternatively add 30 μl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. You should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.
== Induction of Luciferase by Toluene and its Derivatives ==
== Induction of Luciferase by Toluene and its Derivatives ==

Revision as of 09:19, 20 July 2007

Glasgow Main Page

Contents

LB Broth and LB Agar

Transforming BioBricks

  1. Split the competent E. coli cells (~75 µl) in two.
  2. Competent cells (~33 µl) into eppendorfs on ice.
  3. Add 1µl of plasmid DNA.
  4. Incubate on ice for at least 5 mins.
  5. Heat shock +42ºC for 45 secs.
  6. Back on ice for 2 mins to reduce damage.
  7. Add 1ml of soc media.
  8. Incubate +37ºC in a shaker for 1 hour.
  9. Spread the reaction on kan/carb LB plates.
  10. Grow +37ºC overnight.

Minimal Media and Trace Elements

Trace Elements

  1. In 800 ml dH2O dissolve 5 g Na2 EDTA and convert to pH7.
  2. Add the following in order, correcting to pH7 after each.
    • FeCl3 (.H2O) 0.5 (0.83 g)
    • ZnCl2 0.05 g
    • CuCl2 0.01 g
    • CoCl2.6H20 0.01 g
    • H3BO3 0.01 g
    • MnCl2.6H2O (.4H2O) 1.6 g (1.35 g)
  3. Make up to 1 litre, autoclave and store at 4 °C.

M9

  1. Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds
    • 6 g Na2HPO4
    • 3 g KH2PO4
    • 0.5 g NaCl
  2. The add
    • 1 ml 1M MgSO4
    • 200 µl 1M CaCl2
    • 1 ml Thiamine (40mg ml-1 stock)
    • 10 ml Trace Elements
  3. Also add as necessary
    • 15 ml (13C-) Glucose (20% stock) (gives 0.3% final)
    • 1 g (15) NH4Cl (if using M9-)

Tips

  1. Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction.

Primer Design

Qiagen Minipreps

This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.

Procedure

  1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube.

Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

  1. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
  2. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times.

To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.

  1. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
  2. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
  3. Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results.
  4. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.
  5. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Spinning for 60 seconds produces good results.
  6. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.

  1. 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 each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. If you are concerned about the concentration of the DNA, you can alternatively add 30 μl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. You should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.

Induction of Luciferase by Toluene and its Derivatives

(adapted from Willardson et al., 1998)

  1. Grow single colonies of DH5α cells containing pGLTUR in 10 ml LB medium containing 100 µM carbenicillin at 37ºC until OD600 is between 0.2 and 1.0.
  2. Dilute to an OD600 of 0.2 with LB medium.
  3. Induce luciferase transcription by mixing 0.9 ml of the diluted culture with 0.9 ml of LB medium containing increasing amounts of toluene or its derivative compounds.
  4. Incubate in 2.0 ml glass vials (sealed with Teflon septa to avoid loss of the volatile organic compound) at 37ºC for 30 min and then cool on ice for 10 min.
  5. Measure luciferase activity
  6. 75 µl of the induced culture is lysed by addition of 25 µl of 4x lysis buffer (100 mM Tris.HCl [pH 7.8], 32 mM NaH2PO4, 8mM dithiothreitol, 8 mM CDTA, 4% [vol/vol} Triton-X 100, and 200 µg of polymyxin B sulphate per ml).
  7. 25 µl of the lysed cell solution was added to 25 µl of a combined 4x concentrate of luciferase substrates A and B (Analytical Luminescence Laboratories) to give a final 2x concentration of each substrate.
  8. Luminesence read immediately after substrate addition for 45 s in a Turner TD-20e luminometer.
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