Team:Amsterdam/Notebook/Protocols/Miniprepping

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==Miniprepping==
 
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After you have transformed a part from one of our distributions you will want to miniprep it so we have provided the protocol that is used at iGEM HQ. Here at iGEM we use [http://www1.qiagen.com/Products/Plasmid/QIAprepMiniprepSystem/QIAprepSpinMiniprepKit.aspx Qiagen Spin Miniprep Kits] for doing small batches of minipreps.
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=Miniprep=
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==Overview==
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We noticed purification using a [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf miniprep kit] yields insufficient concentrations of DNA, therefore we adapted our own protocol using the reagents supplied with the GeneJET™ Plasmid Miniprep Kit. We noticed that our optimized purification protocol yields dramatically increased concentrations of DNA.
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Please note that while we could have prepared our own reagents we decided it was easier to just use the reagents supplied with the Fermentas miniprep kit.
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<br>
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<br>
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===Protocol===
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==Reagents==
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We used the following reagents from the Fermentas miniprep kit:
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*Resuspension buffer
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*Lysis buffer
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*Neutralization buffer
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*Elution buffer<br/><br/>
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Other reagents:
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*Isopropanol
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*70% ethanol<br/><br/>
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See [http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1027678_HB_QP_0504_WW_LR.pdf here] or [http://www1.qiagen.com/literature/protocols/QIAprepMiniprep.aspx here] for the handbook for the Qiagen Spin Miniprep Kit.  If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information.  The following has been reproduced from the handbook and annotated based on experience with the kit.
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<html>
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Notes:
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<ul type="disc"> <li>Check if the RNase A solution has been added to the resuspension solution.</li>
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<li>Keep the resuspension solution cold.</li>
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<li>Check lysis and Neutralization buffer for salt precipitation.</li>
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<li>All centrifugations should be carried out at >12000 g</li>
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<li>Use 1-5 ml E.coli culture in LB media for purification of high copy plasmids.</li></ul>
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<br>
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<h2>Protocol</h2>
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<ol>
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<li>Pellet 1-5 ml of culture by centrifuging 5 minutes in 2ml Eppendorf tube. If the volume of the culture exceeds 2 ml, first pellet 2 ml, decant supernatant, apply the rest of the culture to the eppendorf tube and centrifuge again.</li>
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<li>Resuspend the pelleted cells in <b>250 µl</b> of the <b>resuspension solution</b>. Pipeting up and down till the cells are completely resuspended. (There is no pellet left).</li>
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'''Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge'''
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<i>Note: Ensure RNase A has been added to the resuspension buffer.</i>
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This protocol is designed for purification of up to 20 &mu;g of high-copy plasmid DNA from
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<li>Add <b>250 µl</b> of the <b>lysis solution</b> and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.</li>
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1–5 ml overnight cultures of ''E. coli'' in LB (Luria-Bertani) medium. For purification of
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low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using
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other methods, refer to the recommendations on page 37.
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Please read “Important Notes” on pages 19–21 before starting.
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Note: All protocol steps should be carried out at room temperature.
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Procedure
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<i>Note: Do not vortex, do not incubate for more than 5 minutes.</i>
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#Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 &deg;C) and transfer to a microcentrifuge tube.<br> Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
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<li>Add <b>350 µl</b> of the <b>neutralization solution</b> and mix immediately and thoroughly by inverting 4-6 times.</li>
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#Add 250 &mu;l Buffer P2 and gently invert the tube 4–6 times to mix.<br> 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.
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#Add 350 &mu;l Buffer N3 and invert the tube immediately but gently 4–6 times. <br> To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
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#Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.<br> A compact white pellet will form.
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#Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
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#Centrifuge for 30–60 s. Discard the flow-through. <br> ''Spinning for 60 seconds produces good results.''
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#(Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.<br>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 DH5&alpha;™ do not require this additional wash step.<br>''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.''
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#Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. <br> ''Spinning for 60 seconds produces good results.''
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#Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.<br> 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.''
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#Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 &mu;l 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.<br>''If you are concerned about the concentration of the DNA, you can alternatively add 30 &mu;L 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.  See notes below for why 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.''
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===Notes===
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<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.</li>
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*Heating the elution buffer to 55&deg;C prior to loading on the column can slightly increase yields.
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<li>Decadent the supernatant in a new 1,5 ml tube.</li>
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*Similarly, doing the elution in two steps (first a 30 &mu;L elution and then a 20 &mu;L dilution) can also slightly increase yields.
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<li>Add <b>650 µl</b> of <b>isopropanol</b>. Invert 4-6 times.</li>
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===Materials===
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<li>Centrifuge for 5 min.</li>
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Do not autoclave solutions containing isopropanol or MOPS; use sterile filtration if necessary.
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<li>Remove isopropanol by decanting.</li>
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Buffer P1
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<li>Add <b>500 µl</b> of <b>70% ethanol</b>, and remove by decanting.</li>
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* 50 mM Tris-HCl pH 8.0
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<li>Let the pellet dry. You can remove some residual ethanol by pippeting.</li>
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* 10 mM EDTA
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* 100 &mu;g/ml RNaseA
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The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).
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Note: It is essential all the ethanol is removed.  
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Buffer P2
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<li>Resuspend the pellet in <b>50 µl</b> water or Elution buffer.</li>
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* 200 mM NaOH
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* 1% SDS
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Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)
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<i>Note: This is just a tris-buffer so it will not interfere with the ligation.</i>
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* 3.0 M potassium acetate pH 5.5
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Buffer N3
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<li>Check the concentration of the plasmids on agarose gel or with a Nanodrop.</li></ol>
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* 4.2 M Gu-HCl
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<br>
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* 0.9 M potassium acetate
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<br>
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* pH 4.8
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Buffer PB
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<br>
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* 5 M Gu-HCl
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</html>
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* 30% ethanol
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*(maybe add 10mM Tris-HCL PH 6.6, and that is better)
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Buffer PE
 
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* 10 mM Tris-HCl pH 7.5
 
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* 80% ethanol
 
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Buffer QBT equilibration buffer
 
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* 750 mM NaCl
 
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* 50 mM MOPS pH 7.0
 
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* 15% isopropanol
 
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* 0.15% triton X-100
 
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Buffer QC wash buffer
 
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* 1.0M NaCl
 
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* 50 mM MOPS pH 7.0
 
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* 15% isopropanol
 
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Buffer QF elution buffer
 
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* 1.25M NaCl
 
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* 50 mM Tris-HCl pH 8.5
 
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* 15% isopropanol
 
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Buffer QN
 
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* 1.6M NaCl
 
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* 50 mM MOPS pH 7.0
 
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* 15% isopropanol
 
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Buffer FWB2
 
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* 1M potassium acetate, pH 5.0
 
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(Source: [http://methodsandreagents.pbwiki.com/], US Patent 6,383,393)
 
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The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3
 
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Latest revision as of 23:59, 21 September 2011

Miniprep

Overview

We noticed purification using a [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf miniprep kit] yields insufficient concentrations of DNA, therefore we adapted our own protocol using the reagents supplied with the GeneJET™ Plasmid Miniprep Kit. We noticed that our optimized purification protocol yields dramatically increased concentrations of DNA. Please note that while we could have prepared our own reagents we decided it was easier to just use the reagents supplied with the Fermentas miniprep kit.

Reagents

We used the following reagents from the Fermentas miniprep kit:

  • Resuspension buffer
  • Lysis buffer
  • Neutralization buffer
  • Elution buffer

Other reagents:

  • Isopropanol
  • 70% ethanol

Notes:

  • Check if the RNase A solution has been added to the resuspension solution.
  • Keep the resuspension solution cold.
  • Check lysis and Neutralization buffer for salt precipitation.
  • All centrifugations should be carried out at >12000 g
  • Use 1-5 ml E.coli culture in LB media for purification of high copy plasmids.

Protocol

  1. Pellet 1-5 ml of culture by centrifuging 5 minutes in 2ml Eppendorf tube. If the volume of the culture exceeds 2 ml, first pellet 2 ml, decant supernatant, apply the rest of the culture to the eppendorf tube and centrifuge again.
  2. Resuspend the pelleted cells in 250 µl of the resuspension solution. Pipeting up and down till the cells are completely resuspended. (There is no pellet left).
    3. Note: Ensure RNase A has been added to the resuspension buffer.
  3. Add 250 µl of the lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
    4. Note: Do not vortex, do not incubate for more than 5 minutes.
  4. Add 350 µl of the neutralization solution and mix immediately and thoroughly by inverting 4-6 times.
  5. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
  6. Decadent the supernatant in a new 1,5 ml tube.
  7. Add 650 µl of isopropanol. Invert 4-6 times.
  8. Centrifuge for 5 min.
  9. Remove isopropanol by decanting.
  10. Add 500 µl of 70% ethanol, and remove by decanting.
  11. Let the pellet dry. You can remove some residual ethanol by pippeting.
    12. Note: It is essential all the ethanol is removed.
  12. Resuspend the pellet in 50 µl water or Elution buffer.
    13. Note: This is just a tris-buffer so it will not interfere with the ligation.
  13. Check the concentration of the plasmids on agarose gel or with a Nanodrop.



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