20 Expression Cloning with Xenopus Oocytes
315
9. The pellet is resuspended in 15).!1 of water. 1 ).11 is used to measure the
RNA concentration spectrophotometrically at OD260 and 1-2 ).11 is run
through a 1% agarose formaldehyde RNA ge1, to check for integrity of
the transcripts, with the rest being used for injection.
10. Thein vitro transcribed RNA (cRNA) is directly injected into oocytes
and after 1-6 days post injection, the protein (transport) assay can be
measured (as described above).
Troubleshooting
Upon successfu1 construction of a re1ative1y 1arge cDNA library (>50,000
clones from fractionated mRNA), containing sufficiently 1arge cDNA inserts (> 1.5kb, checked on an agarose minige1), the 1ibrary is divided into
poo1s of 1000 co1onies (more clones may diffuse the signa1). Each p1ate
of 1000 co1onies is screened for expression ( as described above) and
once a positive poo1 of p1asmids is found, the bacteria containing these p1asmids are subdivided into smaller poo1s by one order of magnitude. These
new poo1s are screened, as weil as the master p1ate and then further subdivided by sib se1ection unti1 a single clone, responsib1e for the increased
expression in oocytes, is found. This part of the work can be very time consuming (several months) and is not always straightforward.
In order to simp1ify the work, sib se1ection is used to narrow down the
positive p1ates and eventually find the positive clone. It is performed the
following way: Assuming you want to screen 25 p1ates of 100 co1onies
each, the way you wou1d do this is to p1ace the p1ates into a grid of 5 x
5 p1ates, with 5 co1umns and 5 rows. The co1umns you can denote A, B,
C, D, E and the rows F, G, H, I, J. Now co1umn A will be made up of p1ates
1-5, co1umn B of p1ates 6-10, co1umn C of p1ates 11-15, etc. The products of
rows and co1umns can now be poo1ed, either at the p1asmid iso1ation 1eve1,
or at the 1eve1 of in vitro transcription, thus reducing the number of reactions from 25 to 10 groups. In addition, once a positive group is found (1et's
say co1umn A and row F), then you can exactly pin -point the positive p1ate in
one step, by 1ooking at the positive co1umn versus its intersecting positive
row (in this case it would be p1ate 1). This sib se1ection technique can be
app1ied at the sing1e co1ony step or at the steps with tens or hundreds of
co1onies per p1ate.
Generally, poo1s of bacteria are on1y subdivided after the protein assay
resu1ts are consistently positive (2 or more times). Typical expressed uptakes for different groups of co1onies during screening are: master p1ates
315
9. The pellet is resuspended in 15).!1 of water. 1 ).11 is used to measure the
RNA concentration spectrophotometrically at OD260 and 1-2 ).11 is run
through a 1% agarose formaldehyde RNA ge1, to check for integrity of
the transcripts, with the rest being used for injection.
10. Thein vitro transcribed RNA (cRNA) is directly injected into oocytes
and after 1-6 days post injection, the protein (transport) assay can be
measured (as described above).
Troubleshooting
Upon successfu1 construction of a re1ative1y 1arge cDNA library (>50,000
clones from fractionated mRNA), containing sufficiently 1arge cDNA inserts (> 1.5kb, checked on an agarose minige1), the 1ibrary is divided into
poo1s of 1000 co1onies (more clones may diffuse the signa1). Each p1ate
of 1000 co1onies is screened for expression ( as described above) and
once a positive poo1 of p1asmids is found, the bacteria containing these p1asmids are subdivided into smaller poo1s by one order of magnitude. These
new poo1s are screened, as weil as the master p1ate and then further subdivided by sib se1ection unti1 a single clone, responsib1e for the increased
expression in oocytes, is found. This part of the work can be very time consuming (several months) and is not always straightforward.
In order to simp1ify the work, sib se1ection is used to narrow down the
positive p1ates and eventually find the positive clone. It is performed the
following way: Assuming you want to screen 25 p1ates of 100 co1onies
each, the way you wou1d do this is to p1ace the p1ates into a grid of 5 x
5 p1ates, with 5 co1umns and 5 rows. The co1umns you can denote A, B,
C, D, E and the rows F, G, H, I, J. Now co1umn A will be made up of p1ates
1-5, co1umn B of p1ates 6-10, co1umn C of p1ates 11-15, etc. The products of
rows and co1umns can now be poo1ed, either at the p1asmid iso1ation 1eve1,
or at the 1eve1 of in vitro transcription, thus reducing the number of reactions from 25 to 10 groups. In addition, once a positive group is found (1et's
say co1umn A and row F), then you can exactly pin -point the positive p1ate in
one step, by 1ooking at the positive co1umn versus its intersecting positive
row (in this case it would be p1ate 1). This sib se1ection technique can be
app1ied at the sing1e co1ony step or at the steps with tens or hundreds of
co1onies per p1ate.
Generally, poo1s of bacteria are on1y subdivided after the protein assay
resu1ts are consistently positive (2 or more times). Typical expressed uptakes for different groups of co1onies during screening are: master p1ates