Smolke:Protocols/Insert prep

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(Protocol)
 
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*Prepare 5 x 100-ul PCR reactions for each insert
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==Protocol==
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*Prepare 5 x 100-ul PCR reactions for each insert (see notes below on reaction volume)
**400 ul water
**400 ul water
**50 ul 10x Taq buffer
**50 ul 10x Taq buffer
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**16 ul 5 mM MgCl2
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**16 ul 50 mM MgCl2
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**10 ul dNTP
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**10 ul 10 mM dNTP
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**10 ul Fwd primer
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**10 ul 10 uM Fwd primer
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**10 ul Rev primer
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**10 ul 10 uM Rev primer
**250 ng plasmid
**250 ng plasmid
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**10 ul 1:10 Taq DNA polymerase
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**10 ul 1:10 Taq DNA polymerase (Starting Mar 2013, we will use 5 ul of 1:20 Taq DNA polymerase)- YHW
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*Taq amplifies at 1000-2000 bp/min, so adjust extension time accordingly
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*After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length
*After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length
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*DpnI treat the PCR products to remove the template - add DpnI directly to the PCR reaction (0.5uL in a 50uL reaction is plenty). Incubate 1hr at 37C. -Josh
*Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
*Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
*Resuspend inserts to appropriate volume for digestion reaction
*Resuspend inserts to appropriate volume for digestion reaction
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*After digestion, gel extract insert to remove the plasmid template
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==Notes==
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*Amplification rates:
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**Taq: 0.5-1 min/kb (YC). 1 min/kb (JKM)
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**Pfu: 2 min/kb (JKM)
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**PfuUltra: 1 min/kb (JKM)
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**PfuUltraII: 0.25 min/kb (JKM)
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**MutazymeII: 2 min/kb (JKM)
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*Size: I consider this ''massively'' oversized. In my experience, 50uL of PCR products (~3ug product) is more than sufficient for cloning. -Josh
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**I think this depends on what you're cloning.  My inserts tend to be <150 bp, which means getting 3 ug of product requires significantly larger PCR volumes.  Smaller inserts are also more difficult to purify without loss downstream, hence the large volume that I generally use. --YC
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***Yeah, but small inserts also means enormous molar ratios even for small masses of DNA. Personally, if I'm working with something so small that I worry about purification, I'd just order the insert as complimentary oligos and avoid the PCR/purification process all together. -Josh
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****Ordering complimentary oligos would certainly be the least labor intensive.  It becomes tricky when the insert is too long for accurate synthesis, which is usually the case with the miRNA and ribozyme switch sequences we typically use. --YC
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*Polymerase: I use Pfu (>500bp) or PfuUltraII (>~3kb) for any lengthy PCR that I intend to clone. -Josh
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*Magnesium: I have never felt a need to vary my magnesium concentration on a reaction-by-reaction basis. So I mix my Taq buffer using a standard amount of magnesium (2mM, personally), and save an extra step. -Josh

Current revision

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Protocol

  • Prepare 5 x 100-ul PCR reactions for each insert (see notes below on reaction volume)
    • 400 ul water
    • 50 ul 10x Taq buffer
    • 16 ul 50 mM MgCl2
    • 10 ul 10 mM dNTP
    • 10 ul 10 uM Fwd primer
    • 10 ul 10 uM Rev primer
    • 250 ng plasmid
    • 10 ul 1:10 Taq DNA polymerase (Starting Mar 2013, we will use 5 ul of 1:20 Taq DNA polymerase)- YHW
  • After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length
  • DpnI treat the PCR products to remove the template - add DpnI directly to the PCR reaction (0.5uL in a 50uL reaction is plenty). Incubate 1hr at 37C. -Josh
  • Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
  • Resuspend inserts to appropriate volume for digestion reaction
  • After digestion, gel extract insert to remove the plasmid template

Notes

  • Amplification rates:
    • Taq: 0.5-1 min/kb (YC). 1 min/kb (JKM)
    • Pfu: 2 min/kb (JKM)
    • PfuUltra: 1 min/kb (JKM)
    • PfuUltraII: 0.25 min/kb (JKM)
    • MutazymeII: 2 min/kb (JKM)
  • Size: I consider this massively oversized. In my experience, 50uL of PCR products (~3ug product) is more than sufficient for cloning. -Josh
    • I think this depends on what you're cloning. My inserts tend to be <150 bp, which means getting 3 ug of product requires significantly larger PCR volumes. Smaller inserts are also more difficult to purify without loss downstream, hence the large volume that I generally use. --YC
      • Yeah, but small inserts also means enormous molar ratios even for small masses of DNA. Personally, if I'm working with something so small that I worry about purification, I'd just order the insert as complimentary oligos and avoid the PCR/purification process all together. -Josh
        • Ordering complimentary oligos would certainly be the least labor intensive. It becomes tricky when the insert is too long for accurate synthesis, which is usually the case with the miRNA and ribozyme switch sequences we typically use. --YC
  • Polymerase: I use Pfu (>500bp) or PfuUltraII (>~3kb) for any lengthy PCR that I intend to clone. -Josh
  • Magnesium: I have never felt a need to vary my magnesium concentration on a reaction-by-reaction basis. So I mix my Taq buffer using a standard amount of magnesium (2mM, personally), and save an extra step. -Josh
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