Clemson University Genomics Institute

M Sodium Phosphate, pH7.2
134 g Na₂HPO₄.7H₂O in 850 ml H₂O
pH to 7.2 with 4 ml 85% H₃PO₄
adjust to 1 L with H₂O

Hybridization solution (1% BSA, 1 mM EDTA, 7% SDS, 0.25M sodium phosphate)
500 ml 0.5 M sodium phosphate, pH7.2
10g BSA(Fraction V, Sigma)
70g SDS
2 ml 0.5 M EDTA, pH8.0
add H₂O to make final 1000 ml

Prehybridization
Roll BAC filters and insert into a 300 ml hyb bottle. Add hyb solution, 30 ml per filter, and allow prehybridization at 65 degrees C for overnight when using new filters. Used filters may be prehybed for 3 hours and the probe added the same day.

Hybridization
Roll BAC filters and insert into a 300 ml hyb bottle. Add hyb solution, 30 ml per filter, and allow prehybridization at 65 degrees C for overnight when using new filters. Used filters may be prehybed for 3 hours and the probe added the same day.

Washing
Remove hyb solution and rinse filters with 200 ml room temperature 1X SSC, 0.1% SDS. Discard the washing solution and transfer the filters to a large tub. Add 2L 65 C preheated 1X SSC, 0.1% SDS and continue washing at 65 C with gentle shaking for 1 hr. Further wash with 0.5X SSC, 0.1% SDS at 65 C if higher stringency is desired. Please be aware that you do need a little bit of background to help in identifying hit addresses and therefore we do not prefer washing too stringently. In most cases, one or two washes with 1X SSC, 0.1% SDS is sufficient.

Autoradiography
Mark the fields of the filters with radioactive ink. Place filters in plastic bags and expose to phosphoimager screens or X-Ray films for 6 hr to overnight.

Comments on Probe Preparation
The probes need to be vector sequence free to avoid high background signal. We use overgo probes for high-throughput library hybridization screening. The temperature for hybridization and washing is 60 C when overgo probes are used.

Stripping BAC colony filters
Wash filters for 10 min at room temp with gentle shaking in 100 mM NaOH, 10 mM EDTA, 0.1% SDS. If necessary, repeat this step until no signal is detected. Remove stripping solution by rinse with ddH₂0 and then wash in 5X SSC for 10 min. Repeat another wash in 5X SSC. The filters are now ready for another hyb or can be dried in 55C oven for 4 hr followed by storage at room temperature.

Plant High Molecular Weight DNA Isolation in Plugs

1. Grind plant tissue in liquid nitrogen and transfer to 1 L flask containing prechilled NIB (on ice). Use about 10 ml NIB per 1 g tissue. Do not put more than 500 ml NIB per flask. We use about 60 to 100 g tissue and divide between 2 flasks.
2. Leave on ice for 6 to 8 min and swirl every 2 min.
3. Filter through 2 layers of cheesecloth and 2 layers of miracloth into clean flasks. Add 1/20 vol. lysis buffer (25 ml/ 500 ml NIB) and leave on ice for 10 min, swirling every 2 min.
4. Aliquot into 50 ml falcon tubes and spin down nuclei using a table top centrifuge with a swinging bucket rotor at 3,200 rpm for 12 to 15 min.
5. Very gently pour off about 45 ml of supernatant and resuspend nuclei with a paintbrush. Consolidate nuclei into ½ the number of falcon tubes with fresh NIB and centrifuge again. Repeat this procedure 2 to 3 more times resulting in the consolidation of nuclei in 1 falcon tube.
6. Remove supernatant from the final consolidation very gently with a large pipetter leaving the nuclei suspended in a final volume of about 2 to 3 ml NIB. Resuspend with paint brush and prewarm nuclei at 45 degrees Celsius for 7 min before adding equal volume of 1.5% low-melting temp agarose in NIB buffer without BME (also equilibrated to 45C). Mix nuclei and agarose gently by tapping tube.
7. Leave falcon tube containing the nuclei-agarose mixture at 45C while filling plug molds to avoid solidification of the agarose. Fill the 75 to 100 ul plug molds (Bio-Rad Corp.) with a 1 ml pipetter (w/ cut off tip) and allow the agarose to solidify at RT or on ice (30 to 60 min). Re-use plug molds by washing/UV sterilizing and use regular lab tape for the back side.
8. Plugs can be removed from molds by forcing them out with air expelled from a eyedropper nipple placed on the back side of the mold. Expel the plugs into a 50 ml falcon tube containing EPS buffer (about 10 vol EPS per plug or 1 ml per plug).
9. Add 1 mg proteinase K per 1 ml EPS and incubate in a hyb oven with moderate rotation at 50C for 18 to 24 hr. Repeat this for another 18 to 24 hr hr with fresh EPS and Proteinase K.
10. Remove EPS with a Falcon tube strainer and add TE (10:1) and PMSF (final concentration of 1 mM). Shake moderately at RT on an orbital shaker for 1 hr and repeat wash with fresh TE and PMSF.
11. Wash plugs 3 times with shaking (1 hr per wash) in TE (10:1) or sterile ddH₂0 to remove residual PMSF. Plugs are now ready for testing and cloning and may be stored safely at 4C in TE (10:1) during periods of cloning. For long term storage, use 0.5 M EDTA or 70% Ethanol. Plugs in 70% ethanol can be placed at -20C after equilibration for the best long term storage option.
12. For HMW DNA restriction, a selected number of plugs are chopped-emaciated immediately prior to use with a razor blade on a microscope slide. To ease pipetting of chopped plugs, add TE or sterile ddH20 as needed. Typically, for about 8 plugs, 400 to 500 ul of TE is added to facilitate pipetting (using cut-off tips).

Recipes
2.25 L 3.25 L
Nuclei Isolation Buffer (NIB)
10 mM Tris 22.5 ml 32.5 ml 1 M Tris
10 mM EDTA 45 ml 65 ml 0.5 M EDTA
100 mM KCl 16.8 g 24.2 g
500 mM sucrose 385 g 556 g
4 mM spermidine 2.3 g 3.3 g
1 mM spermine 0.78 g 1.13 g
Add 0.1% B-mercaptoethanol (BME) immediately prior to use
Bring to volume with ddH₂0

High phenolic tissues add:
PVP MW 40,000 (2% w/v) 45 g
Immediately prior to use add:
DIECA 0.13% 1.3 g/L
Ascorbic acid 0.1% 1.0 g/L
BME 0.2% 2.0 ml/L

It is not necessary to sterilize NIB.

Lysis Buffer
10% Triton-X in NIB

EPS Buffer
0.5 M EDTA pH 9.2
1.0% sarcosyl

EST data processed at CUGI utilizes publicly available software incorporated in a fully automated in-house developed script (ProcEST). The processing occurs in three stages:

Stage I: Trace File Processing
Sequence trace files are converted into fasta files and a quality score files using the phred (Ewing et al, 1998) base-calling program. Vector and host contamination are identified and masked using the sequence comparison program cross_match (Gordon, et al, 1998). Vector trimming excises the longest non-masked sequence and further trimming removes low quality bases (less than phred score 20) at both ends of a read. Sequences are discarded if they have, greater than 5% ambiguous bases, more than 40 PolyA or Poly T bases or less than 100 high quality bases (minimum phred score of 20). At this stage of processing the script generates an overall summary report file, clone report tables, a Genbank submission file and fasta formatted library files of the high quality trimmed sequences and associated quality values. The fasta library is further filtered to remove reads having significant similarity with the species specific mitochondial, rRNA, tRNA or snoRNA sequences downloaded from the Genbank nucleotide database.

Stage II: Assembly of High Quality Sequences
In stage II processing, the filtered library file is assembled using the contig assembly program CAP3 (Huang and Madan, 1999). More stringent parameters (- p 90. -d 60) are typically used to prevent over assembly and help identify potential paralogs.

Stage III: Annotation
Annotation consists of pairwise comparison of both the filtered library and the contig consensus library file against the Genbank nr protein database using BLAST (Altschul et al, 1990 ). The most significant matches (EXP < 1e -9) for each contig and individual clones in the library are recorded. The script generates a web page which displays the best protein match for each contig and singleton. The unigene data set is derived by selecting the clone that best represents the contig, and singletons that have either unique protein matches (EXP < 1e-9) or no known significant matches. The sequence, assembly and homology data are stored in the CUGI database (CUGIdb), facilitating efficient data querying and display. Users can view contig assembly, clones and annotation, download the library and unigene sequence libraries and search their sequences against the EST database using our BLAST/FASTA server facility. Where libraries are unamplified or unnormalized, statistical programs such as SPLUS can be used to analyze differential expression and highlight interesting patterns of expression in the organism under study.

Additional computational analysis will include identifying potential physical markers by mapping the est data to available genomic survey sequences.

References

Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3):403-410.

Ewing, B., Hiller, L., Wendl, M. and Green, P. (1998). Basecalling of automated sequencee traces using phred. I. Accuracy assessment. Genome Research 8, 175-185.

Gordon, D. Abanjian, C., and Green, P. (1998). Consed: A graphical tool for sequence finishing. Genome Research 8, 195-202.

Huang X and Madan A. 1999. CAP3: A DNA sequence assembly program. Genome Res. 9(9):868-877.