User:Anthony Salvagno/Notebook/Research/2009/02/23/Genomic DNA Insertion Day 1: The Cuts

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Contents

Itinerary

  1. Cut pRS413 and yeast genomic DNA with XhoI
  2. Mix pRS413 with CIP to remove phosphate groups
  3. clean plasmid and gDNA with QIAGEN kits

Somewhere in there we are running a gel. I'll write up the protocol las I go.

Last Time Preps

We needed 1.5ug of plasmid DNA, and 2ug of gDNA with appropriate amount of XhoI. So to get the amounts below we are keeping all the ratios the same and using 5ug of both DNAs.

Prep

Gel Prep
Tube Number Vol pRS413 Vol gDNA Vol and Type 10x Buffer Vol BSA 10x Vol XhoI Vol H2O Total Vol
1 33.5uL ---- NE2/8.3uL 8.3uL 3.3 29.5uL 82.5uL
2 ---- 23.4uL NE2/7.5uL 7.5uL 3.75uL 32.75uL 74.9uL

Protocol

  1. Mix enzyme, DNA, buffers, BSA, and water as prescribed above.
  2. Let incubate for 2 hours in 37C dry "bath"
  3. Make a gel
    • We have way more solution this time than last time, so we want to use thicker wells.
  4. After the 2 hours, add 2uL CIP to tube 1 (plasmid) and 3uL of XhoI to tube 2 (gDNA)
    • The CIP will remove the phosphate groups from the ends of the newly linearized plasmid DNA, while the XhoI will boost the digestion process of the gDNA (tough stuff).
  5. Place all pRS DNA into gel
  6. Mix 10uL of gDNA + 2.5uL of dye, and add 10uL of this to gel
  7. Run gel.
  8. Purify remaining gDNA and measure in nanodrop

Results

Gel Results

NanoDrop
Something happened here, where we didn't get any gDNA after purification. The nanodrop reading was 3.3ng/uL. That is bad.

Steve Koch 01:30, 24 February 2009 (EST):Yikes! I have an idea. Which Qiagen kits are you using? The enzymatic cleanup, gel extraction, and PCR kits (or probably any of the affinity-style Qiagen columns do not work well for DNA >4kb or so. Probably most of your gDNA is >4 kb. You have to do things like heat up your elution buffer in order to elute long pieces of DNA. Not in-tune enough with what you're doing to know if that's what happened, but it's definitely affected me in the past. If this is the problem, you should read the manual about the fragment size limitations and / or call tech support. (9 years ago, I used to talk with Kiran Shetty at Qiagen, who was awesome with technical support. One would have to assume, though, that he's moved on to start his own company by now.) Related to this, reading and re-reading the beginning of the Qiagen manual will teach you a lot...possibly about this issue, and definitely about other things that can arise.
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