Picking ES Cell Colonies and Transferring Them to 24-Well Culture Dishes

肖爱华

Picking ES Cell Colonies and Transferring Them to 24-Well Culture Dishes From the Laboratory of Dr. Allan Bradley
! t! p& r6 X/ ~3 a- wBaylor College of Medicine, Houston, Texas
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Picking ES Cell Colonies and
' h8 S" b3 J1 ~4 {Transferring Them to 24-Well Culture Dishes- D8 w$ L  u' _

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( H) u0 C* g( p/ J- }* `+ ]$ f; {        1.        Spot 15 - 25 ul of trypsin in a 24-well array onto the lid % I: A  l' v) _: L; E. v
of a 10 cm plate.
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        2.        After flooding the plate with PBS, pick the colony from
" l+ O5 @5 \- W0 p9 h7 sthe plate using a Pipetman set at 2 ul, and transfer it into the trypsin.  ) _+ u( N/ t6 j" `" |* y1 V+ O/ [3 D/ J
Leave in trypsin for 10-15 minutes.
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4 d& P7 a9 R8 b9 z        3.        Add 20 ul of media from the refed feeder plate to the
! a: j' S  i# k: y& [trypsin spot.  Pipet up and down to break up the colony.% D7 B" A" w' y8 |

& O  k$ \7 \) o3 e" A        4.        Transfer the ES cell suspension (approximately 37 ul) % p, E- Q" g5 u9 A
into the well of a 24-well feeder plate.1 g  ~4 `' T  e0 x( g& M
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        5.        Alternatively, 50% of the cells may be lysed directly : m5 s$ @: a, Y  U, q
for PCR at this point.  Add 15 ul of the cell suspension directly
6 N9 c6 S- D* @# Bto 25 ul of PCR Lysis Buffer.
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From the Laboratory of Dr. Allan Bradley                     
! d* U9 F0 t: hBaylor College of Medicine, Houston, Texas