Kits used for Molecular Biology
PureLink HiPure Plasmid DNA Purification Kit, Midi (Invitrogen, K2100-05)
OBTAIN HIGHLY PURIFIED PLASMID DNA FROM BACTERIA. Kit for 50 rxn.
Before first use:
- use sterilised tips, water etc. for not transfering DNases
- Add 1.5 ml RNase A to 200 ml Resuspention Buffer (R3) and mix well. Store afterwards at 4 °C
- Observe precipitation in Lysis buffer (L7) - if so warm it to 37 °C to dissolve it
Inoculate a overnight culture of E. coli in 25 ml (high copy plasmid) or 100 ml (low copy plasmid) selective LB media in a baffled Erlenmeyer
Centrifuge the cells in a (used) 50 ml conical centrifugation tube for 10 min at 4000 g and discard all supernatant (pipetting)
Add 4 ml Resuspension buffer containing RNase A (R3) and resuspend the cells by pipetting
- in the case of low copy plasmid, use 8 ml Resuspension buffer (R3)
Add 4 ml Lysis buffer (L7) and mix well by inversion/rolling. Do not vortex for not shearing chromosomal DNA
- in the case of low copy plasmid, use 8 ml Lysis buffer (L7)
Incubate for 5 min at RT. Do not incubate for a longer time as this may result in plasmid DNA fragemntation
Add 4 ml Precipitation buffer (N3) and mix well by inversion/rolling. Do not vortex
Centrifuge for 15 min at 12,000 g at RT (not at 4 °C)
- if supernatant is not clear, incubate tube for 5 min, transfer supernatant into a new tube and centrifuge for 5 min at 14,000 g
Column Activation
- add 10 ml Equilibration buffer (EQ1) and let it flow through the column by gravity
- discard flow through
Load the supernatant (pipetting) onto the equilibrated column and let it flow through the column by gravity
- Problem: Column does not fit onto 15 ml conical centrifugation tube
- Do not transfer cell debris onto the column
- Do not let the column let run dry
- Discard flow through
Add 10 ml Wash Buffer (W8) onto the column and let it flow through the column by gravity
- Dicard flow through
Add again 10 ml Wash Buffer (W8) onto the column and let it flow through the column by gravity
- Dicard flow through
Place a new, steril 15 ml conical centrifugation tube under the column
Add 5 ml Elution Buffer (E4) onto the column and let it flow through the column by gravity
Discard the column
Add 3.5 ml Isopropanol and mix well (vortex)
Centrifuge for 45 min at 12,000 g at 4 °C and discard the supernatant
Do not centrifuge at higher forces as the centrifuge tubes will not withstand
Add 3 ml 70% EtOH and mix well (vortex)
Centrifuge for 10 min at 12,000 g at 4 °C and discard the supernatant
Let the pellet air dry (should not smell of EtOH)
Resuspend plasmid DNA in 200 µl TE (~ 1 µg/µl)
Store the DNA at -20 °C
ISOLATE LINEAR DNA FRAGMENTS (50 bp - 10 kbp) FROM ARAGROSE GELS. Kit for 70 rxn.
Before first use:
- Dilute the Wash Solution G with 48 ml 96% EtOH
keep bottle afterwards tightly capped to prevent evaporation of EtOH
- use sterilised tips, water etc. for not transfering DNases
- Verify that the Gel Solubilization solution has not precipitate. Otherwise dissolve precipitate at 37-50 °C
- Preheat Elution solution at 50 °C
- Run the electrophoresis with fresh buffer
- Agarose prepared with TAE can be dissolved more readily than when prepared with TBE
- Expose DNA as short as possible to UV-light (Transilluminator) to reduce DNA-damage
Excise desired DNA band from the gel (scalpel) and transfer it into a Eppendorf-tube
- Cut the band as sharp as possible to have only little excess agarose
- Wear Nitril-gloves when staining DNA with Ethidium bromide
Weight the gel (≤ 300 mg)
Add the triple amount of gel weight of Gel Solubilization solution (≤ 900 µl)
- if the agarose gel is ≥ 2% than use the double amount of Gel Solubilization solution
Dissolve the agarose by incubating it for 10 min @ 50-60 °C
Column Preparation:
- Add 500 µl Column Preparation solution onto the column seated in a 2 ml collection tube
- Centrifuge 1 min at 12,000 g and discard flow through
When the gel is dissolved completely, check that the colour is still yellow
- if the colour is red: add 3 M NaOAc buffer (pH 5.2) in 10 µl steps until the colour changed to yellow
Add the same amount of gel weight of Isopropanol (≤ 300 µl). Mix well
- if the agarose gel is ≥ 2% than use the double amount of Isopropanol
Add the mixture onto the column in 700 µl steps
Same DNA fragments from different lanes can be joined, if you had only few DNA
Centrifuge for 1 min at 12.000 g and discard flow through
Add 700 µl Wash solution G (containing EtOH) and incubate 5 min at RT
Centrifuge for 1 min at 12.000 g and discard flow through
Centrifuge again for 1 min at 12.000 g for eliminating residual Wash solution G
Transfer the column onto a new Collection tube
Add 30 µl preheated Elution solution onto the filter and incubate 1 min
Centrifuge for 1 min at 12.000 g
- if higher quantities are desired (at lower concentration),
Add additional 20 µl preheated Elution solution onto the filter and incubate 1 min
Centrifuge for 1 min at 12.000 g
The Elution solution is 10 mM Tris-HCl, pH 9,0. It does not contain EDTA, thus does not inhibit subsequent reactions (like PCR).
Store the DNA at -20 °C
GenElute Bacterial Genomic DNA Extraction Kit (Sigma-Aldrich, NA1111)
OBTAIN GENOMIC DNA FROM BACTERIA. Kit for 10 rxn.
Before first use:
- add to 5 mg Proteinase K 0.25 ml A. dest. (20 mg/ml).
aliquot and store at -20 °C
- Dilute the Wash Solution with 10 ml 96% EtOH
keep bottle afterwards tightly capped to prevent evaporation of EtOH
- use sterilised tips, water etc. for not transfering DNases
for Gram-positive bacteria only:
- prepare 200 µl of Lysozyme Lysis Solution:
dissolve ~ 10 mg Lysozyme (not provided with the kit) in 200 µl Gram-pos. Lysis Solution and mix well (pipetting)
prepare this solution directly before use
Inoculate a overnight culture of Gram-neg. bacteria strain in 2 ml LB media
Centrifuge cells for 2 min at 12,000 g and discard supernatant
Resuspend cells in 180 µl Lysis Solution T
Add 20 µl RNase A, mix gently (overhead) and incubate 5 min at RT
Add 20 µl Proteinase K Solution, mix gently and incubate 30 min at 55 °C
Add 200 µl Lysis Solution C, mix gently (for not sharing genomic DNA) but well (overhead)
Incubate 10 min at 55 °C
Column Preparation:
- Add 500 µl Column Preparation Solution onto the column seated in a 2 ml collection tube
- Centrifuge 1 min at 12,000 g and discard eluate
Add 200 µl 96% EtOH to the lysate and mix gently (overhead) but well
Transfer lysate onto the binding column using a cutted pipette tip (for not sharing genomic DNA).
Centrifuge 1 min at 7,000 g and discard eluate and replace the collection tube
Add 500 µl Wash Solution 1 onto the column
Centrifuge 1 min at 7,000 g and discard eluate and replace the collection tube
Add 500 µl Wash Solution (containing EtOH) to the column
Centrifuge 3 min at 14,000 g and discard eluate
Centrifuge again 1 min at 14,000 g to eliminate residual EtOH. Discard eluate and replace the collection tube
The collection tubes are several times replaced to avoid to carry over salts
Add 200 µl Elution Solution onto the center of the column and incubate 5 min at RT
Centrifuge 1 min at 7,000 g to elute the DNA
Optional: add another 100 µl of Elution Solution onto the center of the column and incubate 5 min at RT
Centrifuge 1 min at 7,000 g to elute the DNA into the same collection tube
Store the DNA at -20 °C
Troubleshooting:
lysate very gelatinous
-> the sample is too large (less cells should be used next time)
low yield of genomic DNA
-> cells are not lysed completely (increase incubation time with Proteinase K)
-> it was omitted to add EtOH to the lysate
-> lysate/EtOH mixture not homogeneous (mix well although gently by pipetting or inversion)
-> it was omitted to add EtOH to the Wash Solution
-> prior eluation column contained residual EtOH (spin longer or a second time to dry the membrane)
-> DNA elution was incomplete (use Elution Solution, not water. Incubate for 5 min at RT. Repeat elution step)
DNA is sheared
-> DNA was treated improperly. Do not vortex but mix gently (pepetting with a cutted tip or by inversion)
-> cells were old and lyse prematurely
Last modified: 07.05.2010