Plates________________________________
Date_______________
Cassava Field Tissue Collection for Qiagen Kit Extractions:
Use a sampling record, which is a template that is designed according to a racked 8 strip x 12 DNA
extraction tubes to track the samples through the DNA extraction process. We use it to record where
each sample has been placed in the racked tubes during leaf sampling process. Alternatively, the
sample arrangement can be pre-printed and taken to the field for leaf collection.
The sampling papers should be scanned and stored electronically after each collection
Each racked tube-box is pre-labeled with the Sample collection date, plate name, and color-coded using
lab-tapes. In addition to actual writing of the box name, the colored tapes acts as an additional
safeguard against possible plate switching, particularly during extraction steps that require transfer of
supernatants to new sets of tubes.
Sample collection date and method:___xx/xx/xxxx, fresh leaf from field x__________________________________________________
Plate Name:___GS001___________________________________________________________________________________________
Sample information:__Genomic selection seedlings, 2012-2013 season____________________________________________________
1
2
3
4
5
6
7
8
9
10
11
12
A
sample 1
control 1
sample 2
etc
sample 3
etc
B
C
D
sample 4
E
sample 5
F
sample 6
G
sample 7
H
sample 8
Color coding of the day's DNA extraction
Plate 1
Plate 2
Plate 2
Plate 3
1.
2.
BLANK
Extractions will be performed using an 8-tip multichannel pipette working column wise. Therefore we
use strips of 8 tubes and strips of 8 caps. Therefore you might want to layout your field design columnwise.
Put 3mm steel balls into 8-tube strips of Costar Cluster 1.2 ml microdilution tubes (come in Qiagen kit).
You can put balls in 96-well plate to help align them with the strip tubes.
Plates________________________________
Date_______________
Ignore the leaf, that will come
later.
3.
Label all “A” tubes with (12A, 11A, 10A, etc.-careful it is descending order from left to right). Label all
“H” tubes with a line across and the number (1H, 2H, 3H etc.).
4.
Label the plate on the side that faces you when A01 is in the upper left hand corner.
A01
Plate Label
5.
6.
If this is done then you will know that A01 in the top left if you can read the plate name and see the line.
Remember to assign 1 blank in a random place on each plate.
Mark blank tube
Plates________________________________
7.
Date_______________
Fill tubes with a maximum of 0.1 g leaf tissue. For cassava, this is equivalent to the pictured leaf below.
A single, newly expanded leaf per tube is enough for DNA extraction. Adding more tissue will result in
low DNA yield or poor DNA quality!
a. The leaf should be as young as possible.
Just expanded leaf yields large
amount of DNA
b. Roll the leaf and carefully push it into the
tube. Getting the leaf in the tube is a little
tricky, particularly since some genotypes
with the shiny cuticle tend to stick on sides
of the tubes. A small stick may help. Be
careful not to damage the leaf extensively.
8.
If there are contaminates around, such as pollen, then place the strip caps on each column of tissue as it
is harvested.
Plates________________________________
Date_______________
9. Keep samples cool such as on ice or gel packs (4C) until plate is full or samples are able to be frozen.
10. Put breathable tape such as airpore tape over the plate and put in the -80C freezer overnight. Never
allow samples to warm after frozen until lyophilized!
11. Put immediately from freezer into prepared lyophilizer and dry for 48+ hours. When you remove
carefully take off the breathable tape (making sure not to take any leaf with it) and put on the strip caps
(strips of 8) with the end tab on the “A” tubes. Label the caps with the column numbers.
Tab on “A”
tubes and
column
labels.
12. If you are going to extract within the week then grind in the genogrinder (1400 shakes per minute for 2
minutes, check for stuck beads, grind again until all in powder). Store in a -80 freezer until ready for
extraction. Keep these samples frozen and dry or the DNA will degrade!
13. If samples will be stored longer, do not grind, just return to the -80 freezer or store at room temperature
in a vacuum sealed container. Samples stored at RT must be kept dry or DNA will degrade! Grind (step
6) right before extracting. Samples do gain some moisture in the freezer, lyophilizing overnight before
extraction helps with grinding.