User:Jarle Pahr/Kits: Difference between revisions
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{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | {| class="wikitable" border="1" cellpadding="5" cellspacing="0" | ||
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! Plasmid!! Date !! Culture volume !! Antibiotic | ! Strain:Plasmid!! Date !! Culture volume incubated (mL) !! Culture volume centrifuged (mL) !!Medium /Antibiotic !! Incubation vessel !! Incubation time!! OD600 !! Elution volume (uL) !! Obtained DNA concentration (ng/uL) !! Total yield (ug) !! Yield (ug / mL * OD600) !! 260/280 !! 260/230 | ||
|- | |- | ||
|pSB-M1g|| || | |Dh5a:rlig1 (pJP-1)||21.5.13||10 mL||~4.5||LB + Kan 50 ug/mL||50 mL tube||~ 8h||~2.2||50|| 49 || 2,45|| 0,25 || || | ||
|- | |||
|Dh5a:rlig1 (pJP-1)||21.5.13||10 mL||~4.5||LB + Kan 50 ug/mL||50 mL tube||~ 8h||~2.2||50|| 35 || 1,75|| 0,18 || || | |||
|- | |||
|Dh5a||21.5.13|| ||~3.5||LB ||13 mL tube|| || ||2.7 || || || || || | |||
|- | |||
||Dh5a:pSB-M1g||21.5.13|| ||~4||LB + Kan 50 ug/mL ||13 mL tube|| ||2.6 |||| || || || || | |||
|} | |||
DongSheng Bio Eco friendly plasmid extraction kit: | |||
Observed plasmid yields: | |||
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! Strain:Plasmid!! Date !! Culture volume incubated (mL) !! Culture volume centrifuged (mL) !!Medium /Antibiotic !! Incubation vessel !! Incubation time!! OD600 !! Elution volume (uL) !! Obtained DNA concentration (ng/uL) !! Total yield (ug) !! Yield (ug / mL * OD600) !! 260/280 !! 260/230 | |||
|- | |||
|Strain:Plasmid|| Date ||Culture vol inc||Culture vol centr||medium||incub vessel||incub time||OD600||El. vol.|| conc || tot || rel || 260/280 || 260/230 | |||
|} | |} | ||
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Qiaex II: Use a thermomixer (high mixing, RPM ~ 500 ) to get constant mixing during incubation. | Qiaex II: Use a thermomixer (high mixing, RPM ~ 500 ) to get constant mixing during incubation. | ||
In vitro transcription kits: | |||
https://www.neb.com/products/e2050-t7-quick-high-yield-rna-synthesis-kit | |||
Latest revision as of 07:44, 21 May 2013
Commercial kits for applications in molecular biology:
HisExpress protein purification columns: http://www.claremontbio.com/HisExpress_Protein_Purification_Columns_s/72.htm
(available at http://www.bioquote.com/product.php?id=291680)
PrepEase his-purification: http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?navMode=34000&productId=131366&navAction=jump&aId=productsNav#1_1
Affinity chromatography products at Jena Bioscience: http://www.jenabioscience.com/cms/en/1/browse/1450_affinity_chromatography.html?gclid=CJCPmK6HvrUCFQtY3godFWEAtg
| Kit | Use | Advantages | Disadvantages | Time | Experience |
|---|---|---|---|---|---|
| Promega Wizard SV miniprep | DNA isolation | Quick | Low yield | 45 - 60 min | |
| Promega PureYield™ Plasmid Miniprep System | High-purity DNA isolation | ||||
| Machery-Nagel NucleoBond Xtra Midi (midiprep) | DNA isolation | High yield (~20 ug), pure product. | Slow | 2 d | Yields of 100-140 ng/uL in 200 uL, for a total of about 20 ug. Yield may depend on skill in handling washing and resuspension of DNA pellet. Might be more efficient to do several (~10) parallel minipreps instead? |
| Qiaquick gel extraction | DNA extraction from gel | Fast | Impure product | Impurities give absorbption peak at 230 nm in eluted product. | |
| Qiaquick PCR purification | Purification of PCR products | Fast | 15 min | ||
| QIAEX II gel extraction | DNA extraction from gel | Higher purity than qiaquick | Slower than Quiaquick | 1-5 h | Yield of about 200 ng from ~1.5 ug starting amount of plasmid. |
| QIAGEN PCR Cloningplus Kit | Subcloning (TA cloning) | ? | ? | ? | |
| Dongsheng Bio Eco-friendly Plasmid miniprep kit | DNA isolation | ? | - | ||
| Dongsheng Bio PCR and DNA Fragment Purification Kit | Purification of PCR fragments | ? | |||
| QiaAmp |
Notes:
Promega SV Wizard miniprep:
Observed plasmid yields:
| Strain:Plasmid | Date | Culture volume incubated (mL) | Culture volume centrifuged (mL) | Medium /Antibiotic | Incubation vessel | Incubation time | OD600 | Elution volume (uL) | Obtained DNA concentration (ng/uL) | Total yield (ug) | Yield (ug / mL * OD600) | 260/280 | 260/230 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dh5a:rlig1 (pJP-1) | 21.5.13 | 10 mL | ~4.5 | LB + Kan 50 ug/mL | 50 mL tube | ~ 8h | ~2.2 | 50 | 49 | 2,45 | 0,25 | ||
| Dh5a:rlig1 (pJP-1) | 21.5.13 | 10 mL | ~4.5 | LB + Kan 50 ug/mL | 50 mL tube | ~ 8h | ~2.2 | 50 | 35 | 1,75 | 0,18 | ||
| Dh5a | 21.5.13 | ~3.5 | LB | 13 mL tube | 2.7 | ||||||||
| Dh5a:pSB-M1g | 21.5.13 | ~4 | LB + Kan 50 ug/mL | 13 mL tube | 2.6 |
DongSheng Bio Eco friendly plasmid extraction kit:
Observed plasmid yields:
| Strain:Plasmid | Date | Culture volume incubated (mL) | Culture volume centrifuged (mL) | Medium /Antibiotic | Incubation vessel | Incubation time | OD600 | Elution volume (uL) | Obtained DNA concentration (ng/uL) | Total yield (ug) | Yield (ug / mL * OD600) | 260/280 | 260/230 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Strain:Plasmid | Date | Culture vol inc | Culture vol centr | medium | incub vessel | incub time | OD600 | El. vol. | conc | tot | rel | 260/280 | 260/230 |
Qiaquick gel extraction kit:
Qiaex II: Use a thermomixer (high mixing, RPM ~ 500 ) to get constant mixing during incubation.
In vitro transcription kits:
https://www.neb.com/products/e2050-t7-quick-high-yield-rna-synthesis-kit
