LABORATORY PROCEDURES FOR GENOMIC LIBRARIES
REFERENCE NO: GLI/1998/4/4 TITLE: MAKING UP SETS OF DNA AGAROSE PELLETS FOR ISSUE TO USERS Materials Required Tray of the correct DNA-agarose pellets stocks from the 4oC cold cabinet
REFERENCE NO: GLI/1998/4/4
TITLE: MAKING UP SETS OF DNA AGAROSE PELLETS FOR ISSUE TO USERS
Tray of the correct DNA-agarose pellets stocks from the 4oC cold cabinet
See the separate SOP on how to print the labels
Decide how many sets you need to make, and put rows of 40(ICI), 27(ICRF) or 50(MY) micro-tubes in the racks.
Now take stock tube 1(ICI/MY) or A(ICRF) of the DNA agarose stocks. [at present these are in a mixed form in both the ICI and ICRF libraries. The old way was to make plugs and the new way is to make pellets (a.k.a. blobs)]
Put the thinner end of the spatula into the 70% alcohol bottle, remove, and wipe dry with a tissue.
Use the spatula to remove one plug or 2 blobs from the stock tube, and place in each of the tubes labelled 1 or A. Make sure that each tube has a plug/blob in it.
Put the lids on these filled micro-tubes, and move them to the left - so that they are distinguishable from the unfilled tubes.
Put the spatula back in the alcohol and wipe dry. This needs to be done between each stock tube, because the DNA in each is different, and must not be mixed up.
Now tube 2 or B can be done.
When all the microtubes are filled they should now be placed in sets.
Label the appropriate number and size of 'press-seal' polythene bags with either:
ICI Primary Pool Set 1-40
Fill each bag with the correct number of tubes:
ICI 40 tubes labelled 1-40
Amend the stock-taking record by adding on the number of sets that have been made.
REMEMBER 1 PLUG OR 2 BLOBS PER PRIMARY POOL TUBE FOR ICI OR ICRF LIBRARIES
? BLOBS PER PRIMARY POOL TUBE FOR THE MY LIBRARY
This is essentially very similar to the primary sets
ICRF Secondary Pools Primary pool number + 1-20 and the corresponding plate pool numbers
ICI library; make rows of microtubes labelled 1-20, according to how many copies of the super-secondary sets need to be made. Then make columns of microtubes labelled A-I. [ At present the DNA agarose for the plate pools A-I are a mixture of plugs and pellets. If the agarose is a plug, then it will have to be divided into three, and then the number of sets of A-I tubes will have to be in multiples of three. To divide a plug into three, use an alcohol cleaned spatula, lift the plug into the upturned lid of the bijou bottle and use the end of the spatula to cut the plug into three. Each tube will then receive 1/3rd of a plug. This is the equivalent of a 40 Ál pellet]
ICRF library; this is similar to the ICI library, except that you have to determine the plate numbers contained within each pool. At present all the plate pool DNA is present as plugs.
First put the 8 or 9 plate pool tubes in a small labelled 'press-seal' bag, then place this bag within a medium labelled 'press-seal' bag and add the 20 tubes labelled 1-20.
Place the filled bags into their correct sandwich box in the 4oC cold cabinet, placing the new sets under any that are already in the box.
Amend the stock record chart by adding on the number of new sets that you have made
There is a master record sheet of how many pellets remain of the super-rows and super-columns. Please complete this.
Guidelines prepared for CABRI by HGMP, December 1998
© The CABRI Consortium 1999-2013.