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==Plasmid digestion==

J.W. Jahng 2000

*to linearize cDNAs of interest for the preparation of cDNA blot

1. you may want to have three different concentrations of each cDNA on your blot,  like 20ng, 100ng, and 500ng.

2. so you would need 1ug of  each plasmid for 1 set of cDNA blot, and your blotter is big enough to make 2 sets of cDNA blots at a time, so set up the digestion with 2ug of each plasmid.

3. The basic formular for the digestion mixture is
    -----------------------------
    43ul   plasmid + ddH2O
     5ul   10 x buffer
     2ul   restriction enzyme
    -----------------------------

4. My formular for digestion was
  ------------------------------------------------------------------------------------------------------
   Plasmid                          Enzyme                            Buffer                             dH2O
  ------------------------------------------------------------------------------------------------------
   TH (1ug/ul)      2ul         EcoR1        2ul                React 3       5ul              41 ul
   NPY(0.5ug/ul) 4ul         EcoR1        4ul                React 3       5ul              37 ul
   ORX(0.6ug/ul) 3.4ul     EcoR1         2ul               React 3        5ul             39.6 ul
   pBS(1ug/ul)     2ul         EcoR1        2ul                React 3       5ul              41 ul
  ------------------------------------------------------------------------------------------------------
  *your NPY is quite tough and needs more enzyme for digestion, use 4ul instead of 2ul.
  *if you also want to do some other cDNAs like POMC and CRH,  they both work fine 
    with EcoR1 digestion.
  *do you also have AGRP cDNA? I just don’t have any information for AGRP cDNA.

5. when you set up the digestion, take another 0.5 ug of each plasmid into second set of clean tubes and bring up the volume to 12.5 ul with TE, and keep them in the freezer. You’ll load them on a gel with your plasmid digests later on as ‘un-cut control’.

6. Check your digests on a gel.
  *6 hour digestion at 37 C worked fine for me, by the way.
  * take 12.5 ul each of your plasmid digest into clean tubes, then add 3ul of loading dye.
  *of course you want to add the loading dye to your ‘un-cut control’ tubes too.

7. Run the samples on 1 % of agarose gel (0.5 x TBE for gel and buffer).
  *make a medium gel (200ml) with a small comb, and run for 2 h at 130 volts.

*** you can keep your digests in the freezer until you use, but be sure you use them 
       in A week, otherwise better discard them***

[[Category:Protocols]]