APPENDIX I
Extraction buffer (EB)
lOOmM Tris pH 8.0
50mM EDTA pH 8.0
500mM Nacl
lOmM (3-Mercapto ethanol
Homogeneizing buffer (HB)
300mM Sucrose
5mM MgCl2
50mM Tris pH 7.8;
(filter sterilized)
HBT
98ml HB
2ml Triton X-100;
(filter sterilized)
Lysis buffer
50mM Tris pH 8.0
50mM EDTA pH 8.0
50mM Nacl
2% Sarkosyl
TE (Tris-EDTA)
lOmM Tris pH 8.0
ImM EDTA.
1 X TAE
0.04 M Tris-acetate
0.001M EDTA
Adjust pH 8.0 with glacial acetic acid
10 X Gel loading buffer n
10% Bromqpiuenol blue
150mM EDTA
70% glycerol
8.
10 X ligation buffer
400mM Tris.C! pi I 7.5
lOOmM MgCl2
lOOmM DTT "
lOmM ATP
50u.g/ml BSA (to be added only for cloning in phage lambda)
9.
LB Medium
1.0%
0.5%
1.0%
adjust
10.
NaCl
Yeast extract
Bactotryptone
pH6.8 with NaOH.
LB Plates
1.6g Agar powder in 100ml LB medium
11
LB-1XA
0.5mM 1PTG
0.004% X-gal
lOOfXg/ml Ampicillin
Add to LB Agar medium
12.
SOC Medium
LB supplemented with
10mm MgS0 4
10mm MgCl2
20mm Glucose
13.
TB Medium
1% Bactotryptone
0.5% NaCl
pH 6.8
14.
TB Top Agar
0.8% Aear in TB Medium
NZCYM Medium
1% NZ Amine
0.5% NaCl
0.5% Yeast extract
0.1% Casamino Acids
0.2% MgS0 4 .7H : 0
Phage buffer
20mm Tris (pH 7.4)
100mm NaCl
10mm MgSo4.7H20
Denaturing solution
0.5M NaOH
1M Nacl
Neutralizing solution
0.5M Tris pH 7.5
3M Nacl
20 X SSC
3M Nacl
0.3M Sodium citrate
Prehybridizing solution
0.4M Sodium dihydrogen phosphate (pH 6.8)
0.25% SDS
125pg/ml Sonicated salmon sperm DNA
ImM EDTA.
5% Denhardts solution
(100% Denhkrdt's solution is 2% PVP and 2% Fico
10 X TBE
0.045M Tris-borate
0.001M EDTA
APPENDIX1I
The sequences reported in this study \vill appear in the EMBL databank
under the iblUnvine Accession number;
Sequence
Accession No.
B. campestris 5S rDNA
X 60723
B. campestris rDNA intergenic spacer
X 73031
B. jimcea rDNA intergenic spacer
X 73032
APPENDIX III
List of publications:
1.
Bhatia, S.. Singh, K.. Jagannathan, V. and Lakshmikumaran, M.
(1993). Organization and sequence analysis of the 5S rRNA genes
in Brassica campestris. Plant Science 92: 47-55
2.
Singh, K.. Bhatia, S. and Lakshmikumaran, M. (1994). Novel
variants of the 5S rRNA genes in Eruca sativa. Genome 37 (In
Press).
3.
Bhatia, S., Negi, M.S., Jagannathan, V. and Lakshmikumaran, M.
(1994). The shortest ribosomal repetitive DNA element identified
in the intergenic spacer of Brassica nigra. Genome (Submitted).
4.
Jain, A.. Bhatia, S., Banga, S.S., Prakash, S. and Lakshmikumaran.
M. (1994). Potential use of random amplified polymorphic DNA
(RAPD) technique to study the genetic diversity in Indian mustard
(Brassica juncea) and its relationship to heterosis. Theor Appl
Genet (In Press).
5.
Bhatia, S., Jagannathan. V. and Lakshmikumaran.
M.
Characterization and sequence analysis of the rDNA intergenic
spacer (IGS) of B. campestris: Comparisons of the IGS from the
three diploid Brassica species. (In preparation).