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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
Sterile 1.5ml Sarstedt micro-tubes
Printed labels
Tube rack for micro-tubes
5mM EDTA pH8 [see
GLI/1998/3/9]
Eppendorf 12.5ml combitip and dispenser
White plastic spatula
70% alcohol [see
GLI/1998/3/9]
Tissues
Gloves


Printed Labels

See the separate SOP on how to print the labels
You will require the following different sets of labels:

ICI Primary Pools

1-40

ICI Secondary Pools

Primary pool number + 1-20 for pooled rows and columns
and + A-I for plate pools
(e.g.; for Pool 14 it would be 14-01, 14-02, 14-03 ...........14-20 and 14A, 14B ...... 14I)

ICRF Primary Pools

A-Z and ZZ

ICRF Secondary Pools

Primary pool number + 1-20 for pooled rows and columns and the corresponding plate pool numbers
(e.g.; for Pool E it would be E-01, E-02, E-03 ...........E-20 and the plate pool numbers 4X33, 4X34, 4X35 ........ 4X40
[the pool letter is not needed here as each plate has a unique number])

MY Primary Pools

1-50

MY Secondary Pools

Primary pool number + 1-20 for pooled rows and columns and the corresponding plate pool numbers
(e.g.; for Pool 5 it would be 5-01, 5-02, 5-03 ...........5-20 and the plate pool numbers 110, 112, 113, ........ 118
[the pool letter is not needed here as each plate has a unique number. BUT REMEMBER THE PLATE POOLS IN THE LANDER MOUSE LIBRARY ARE NOT ALWAYS CONSECUTIVE, OR WHAT YOU WOULD EXPECT TO BE IN THAT POOL. SO EXTRA CARE IS NEEDED])


Primary Pool Sets

Decide how many sets you need to make, and put rows of 40(ICI), 27(ICRF) or 50(MY) micro-tubes in the racks.
One row = one complete primary pool set
Label the tubes
Wear gloves from now on
Remove the lids from the microtubes and place in a clean large petri dish with lid
Fill the combitip with the 5mM EDTA and set the dispenser to 2 - this will deliver 500 Ál per tube
Fill all the tubes with the EDTA, taking care not to splash or overflow, and remembering to refill the combitip before the EDTA runs out.
It is essential that each tube has EDTA in it (just one tube missed out, will result in that agarose pellet drying out, so that the set will be incomplete for the user)

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.

Etc., Etc.

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
or ICRF Primary Pool Set A-ZZ
or MY Primary Pool Set 1-50

Fill each bag with the correct number of tubes:

ICI 40 tubes labelled 1-40
or ICRF 27 tubes labelled A-ZZ
or MY 50 tubes labelled 1-50


Place the bags in the correct 4oC cold store container, underneath any existing sets, so that the older sets get used up first.

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


Secondary Sets

This is essentially very similar to the primary sets
Labels need to be printed
ICI Secondary Pools Primary pool number + 1-20 and A-I
(e.g.; for Pool 14 it would be 14-01, 14-02, 14-03 ...........14-20 and 14A, 14B ...... 14I)

ICRF Secondary Pools Primary pool number + 1-20 and the corresponding plate pool numbers
(e.g.; for Pool E it would be E-01, E-02, E-03 ...........E-20 and the plate pool numbers 4X33, 4X34, 4X35 ........ 4X40)


MY Secondary Pools Primary pool number + 1-20 for pooled rows and columns and the corresponding plate pool numbers
(e.g.; for Pool 5 it would be 5-01, 5-02, 5-03 ...........5-20 and the plate pool numbers 110, 112, 113, ........ 118

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.

An ICI Secondary set will contain 29 tubes:


20 tubes labelled 1-20
9 tubes labelled A-I

An ICRF Secondary set will contain 28 tubes:


20 tubes labelled 1-20
8 tubes labelled with the plate pool numbers


When all the tubes are filled with pellets/plugs, as described for the primary pool sets then assemble the sets

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.

The bag should be labelled with;




the library name
the pool number
1-20
A-I or 4X33-40 (for example)

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
Page layout by CERDIC
Copyright CABRI, 1998

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This work cannot be reproduced in whole or in part without the express written permission of the CABRI consortium.
Site maintained by Paolo Romano. Last revised on April 2013.