Solutions to prepare ahead of time:

1.5% In-Cert Agarose in SM buffer (make up in 50 ml tube)

250 mM EDTA + 1% SDS

TE buffer (10 mM Tris, I mM EDTA, pH 8.0)

Plug storage (20 mM Tris, 50 mM EDTA, pH 8.0)

 

A freshly prepared solution of Proteinase K (20 mg/ml) is also required.

 

 

1.    Used plug molds are stored in 10% Liquinox.  If you’re using these molds, rinse the molds thoroughly with di water and dry before use.  If you’re using new plug molds, they’re ready to go out of the bag.  Break them apart before using and proceed to step 4.  Do not pass go, do not collect $200.

2.     Seal the bottoms of the molds with scotch tape.  2 molds can fit on one piece of tape (width).

3.    Use a razor blade to carefully cut the tape between the plug molds, making sure that the bottoms of the wells stay sealed with tape.

4.    Using small pieces of labeling tape, label each mold with the sample it is to receive.

5.    Melt the 1.5% InCert agarose in the microwave.  CAUTION: this type of agarose boils VERY quickly.  Initially microwave 5 to 7 seconds.  Then heat for 2-3 second intervals and swirl carefully between heatings until the agarose boils.  Attend the microwave at ALL times during melting.  After the agarose has completely melted, allow to cool briefly.  Have your viral concentrates ready; have the P 1000 set to 425 ul (or 500ul) and the P 200 set to 80 ul.

6.    In a 2 ml Eppendorf tube, add 425 ul (or 500ul) * of viral concentrate and 425 ul (or 500ul) * of the melted 1.5% InCert agarose.  Cap and vortex briefly.  If you are preparing multiple samples simultaneously, don’t forget to label everything, Dumbass.

7.    Carefully dispense 80 ul of the mixture into each well of the appropriately labeled plug mold*.  

8.    Place the molds in the refrigerator (4 deg. C) for about 20 minutes to set.

9.     Set up a tube for each mold:  Pour about 2 ml of 250 mM EDTA + 1% SDS into a labeled 15 ml centrifuge tube.  Add 100 ul of a freshly prepared Proteinase K solution (20 mg/ml).  

10.    Remove the tape from the bottom of the plug mold.  Use a sterile disposable inoculation loop to push the lugs out of the mold and into the tube.  The loop must be cut in half (along the axis of the handle) in order to fit through the wells of the plug mold (see diagram).

11.     Make sure all plugs are in the solution at the bottom of the tube.  Incubate at room temp in the dark (like, put it in a drawer, man) overnight.

12.    The next day, carefully decant the Proteinase K/SDS solution, using a sterile disposable loop to hold back the plugs.  Rinse the plugs 3 times, 30 minutes each, with 10 ml TE buffer.  Use the loop to hold back the plugs while the liquid is carefully decanted each time.  

13.    Store plugs in 4-5 ml 20 mM, Tris 50 mM EDTA, pH 8.0 at 4 degrees C until use.

 

 

 

* Each well of the plug mold will hold 80 ul: 40 ul of virus suspension + 40 ul of 1.5% In-Cert agarose.  A full mold has 10 wells and will require 10X these amounts, or 400 ul of virus suspension + 400 ul of In-Cert.  Because of pipetting error and the viscosity of the agaose, it is necessary to add extra liquid to still end up with 10 full wells, hence 425 ul of virus suspension + 425 ul In-Cert.  If you are making fewer than 10 plugs then scale back your volumes accordingly, but don’t foget to allot extra for the pipetting error.  (note from SB: I have found that if I use 500ul volumes that I will always have enough for 10 plugs and usually only have a small amount left over.)