User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/23

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Library preparation for Alien genes

Confirmation of adapter ligation by PCR

Reaction mixture conc. 1x x4 ' lane Primer set
Template (Adapter ligated AGM fragments, 2 days ligation)2.5 ng/ul0.2511Primer Atitin + Btitin
TopTaq Buffer10x142Primer Atitin
dNTP mix10 mM each0.20.83Primer Btitn
Primer mix5 uM each0.54AV10027
TopTaq5 U/ul0.050.2
Milli Q832
10 ul
PCR cycle
94 C3 min
94 C40 sec
65 C1 min
72 C6 min
x 20 cycle

load 5.0 ul of sample

Image:0121-2.jpg

Template & PCR reaction confirmation

Reaction mixture conc. 1x x3 ' lane Template
Template0.51.51AGM before adapter ligation (10 ng/ul)
TopTaq Buffer10x132AGM after adapte ligation (7.0 ug/ul)
dNTP mix10 mM each0.20.63CCA3 (3.8 ng/ul)
Primer mix (AV10027)5 uM each0.5
TopTaq5 U/ul0.050.15
Milli Q7.7523.25
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min

load 5.0 ul of sample

Image:0123.jpg

Confirmation of adapter ligation by PCR

Reaction mixture conc. 1x x4 ' lane Primer set
Template (Adapter ligated AGM fragments, purified by MinElute)33.8 ng/ul0.2511Primer Atitin + Btitin
TopTaq Buffer10x142Primer Atitin
dNTP mix10 mM each0.20.83Primer Btitn
Primer mix5 uM each0.54AV10027
TopTaq5 U/ul0.050.2
Milli Q832
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min

load 6.0 ul of sample

Image:0127.jpg

Reviving Yeast Cultures Rehydration and Growth of freeze dried cultures

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

Slightly modified protocol.

Revived S. pombe 2722 Added a blank tube on 24June10

Image: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 ampoule 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).

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