User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/05

From OpenWetWare
Jump to: navigation, search
OpenWetWare photo.jpg Alien genes Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Library preparation for Alien genes

Control experiment (library confirmation by PCR)

Reaction mixture conc. 1. 10 kb mix 2. AGM (after gel excision)
Template 2.5 ng/ul 1 5
TopTaq Buffer 10x 1 1
dNTP mix 10 mM each 0.2 0.2
Primer mix (Atitin/Btitn) 5 uM each 1 1
TopTaq 5 U/ul 0.05 0.05
Milli Q 6.75 2.75
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 1 min
x35
72C 5 min

load 5.0 ul of sample

0146.jpg


Reviving Yeast Cultures Rehydration and Growth of freeze dried cultures

http://www.ncyc.co.uk/revive.html

Slightly modified protocol.

Revived remaining colonies

NCYC Culture Strain
1022 cerevisiae
2355 Schizosaccharomyces japonicus
427 Schizosaccharomyces octosporus
204 Saccharomyces pastorianus
415 Saccharomyces bayanus
543 Saccharomyces kluyveri
700 Saccharomyces paradoxus
777 Saccharomyces dairenensis
814 Saccharomyces exiguus
971 Saccharomyces unisporus
2878 Saccharomyces barnettii
2888 Saccharomyces mikatae
2889 Saccharomyces kudriavzevii
2890 Saccharomyces cariocanus
3083 Saccharomyces humaticus
3084 Saccharomyces yakushimaensis
2991 Saccharomyces spencerorum
2307 Saccharomyces servazzii

Yeastamp.gif

Preparation - Wipe down hood with 70% alcohol - Turn on UV light for a min of 15 min - Turn on hood

Each yeast is dispatched as a small quantity of freeze-dried culture sealed under vacuum in glass ampoules. Ampoules should be opened in the following way:

     1). Check the number on the label inside the ampoule.
     2). Score the glass with a suitable file or scoring impliment at the level of the cotton wool plug.
     3). Wipe the ampoule with alcohol.
     4). Apply the end of a red-hot glass rod to the file mark to crack the glass. Leave the glass rod in contact with the ampoule for 2-3 seconds if the ampoule does not crack immediately. If the ampoule still fails to crack repeat the process until successful.
     5). Remove the tip of the ampoule and cotton wool plug and place in a container for subsequent sterilization before disposal.
     6). Using a sterile Pasteur pipette, add about 0.5ml YM broth (Difco 0711-01) or malt extract to the dried material.
     7). Gently resuspend the dried culture and transfer to a culture bottle containing approximately 10ml of the same medium. In order to prevent pH shock etc. it is recommended that no more than 10mls nutrient broth be used for initial growth of the culture.
     8). Unless otherwise indicated, most yeasts should be incubated at 25°C (our model can not go below 29C). Growth may be slow immediately after resuscitation. It may be necessary to incubate cultures for at least 5 days before discarding. Growth is usually stimulated by aeration which may be achieved by shaking or rocking the culture.
     9). The discarded ampule should be placed in a container for subsequent sterilization before disposal.

Growth Media: All NCYC strains (except where indicated) can be routinely grown in YM broth (Difco 0711-01). Other suitable nutrient media include:

   * YM: 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose
   * YEP-glucose: 0.5% yeast extract, 0.5% peptone, 1% glucose
   * Malt Extract: 0.3% malt extract, 0.5% peptone
   * Sabouraud's Glucose: 1% peptone, 4% glucose
   * YPD medium: 1% yeast extract, 2% peptone, 2% glucose
   * Yeast Nitrogen Base (Difco 0392-15-9): a chemically defined medium to which a carbon source must be added.
   * Yeast Nitrogen Base without amino acids (Difco 0919-15-3), which can be supplemented with appropriate amino acids or other source of nitrogen (useful for some genetically defined strains). A carbon source must be added.

Agar when required is added to a final concentration of 1.5 - 2.0% (All percentages are given weight/volume)

More information about these media is given by J P van der Walt & D Yarrow, 'Methods for the isolation, maintenance, classification and identification of yeasts' in 'The Yeasts . A Taxonomic Study', third edition by ed. N J W Kreger-van Rij, Elsevier, (1984) pp 45-104, and in 'Yeasts: Characteristics and Identification', third edition by J A Barnett, R W Payne & D Yarrow, Cambridge University Press, (2000).