cDNA libraries for Genome Project

John Davies jdavies at iastate.edu
Wed Oct 20 10:07:55 EST 1999


We will soon start making the cDNA libraries to be used in the sequencing
project for the genome grant.  I have given some thought to the treatments
that will be used prior to RNA isolation and have included a list of these
treatments below.  I'd like input from you regarding these conditions.  What
other treatments would you like included?

Any input would be greatly appreciated.

Regards,
John


Proposed conditions for RNA isolation:

Light quantity and phototrophic/heterotrophic growth:
phototrophic low light (10 umole/m2 s)
phototrophic med light (100 umol/m2 s)
phototrophic high light (1000 umol/m2 s)
phototrophic high CO2
phototrophic low CO2
phototrophic high CO2 to low CO2 transition (time course)
heterotrophic med light
heterotrophic dark

Nutrient deficiencies; (a time course of RNA isolation will be done for these
conditions):
nitrogen
phosphorous
potassium
sulfur
iron
copper
calcium

Other stresses; (a time course of RNA isolation will be done for these
conditions):
heat (temp?)
cold (temp?)
high salt (conc?)
cadmium
high copper
hydrogen peroxide

Light quality:
blue light
red light
ultra violet

Deflagellation and Mating:
deflagellation recovery time course
gametogenesis time course
zygote formation time course
zygote maturation time course

_____________________________________________________

John Davies
Department of Botany
459 Bessey Hall
Iowa State University
Ames, IA 50011-1020
phone 515-294-4799
fax 515-294-1337

---------


A follow-up to this message, from Arthur Grossman:

Making of the cDNAs

At this point it looks like 21 gr (since it is both NIT1 and
NIT2 plus) will be the strain used for library construction.  It is my
understanding that it is probably not very distantly related to
CC-125.  If there are other ideas about which strain should be used
please let us know.  Also, if your are willing to make RNA from
conditions in which you are interested (other than the ones that
were mentioned in John's note) please let us know... any help will
be appreciated.

Arthur Grossman







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