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BDGP Resources |
For SuPorP, pGT1, and p{Epgy2} elements, see the Bellen lab web site [HERE] Berkeley Drosophila Genome Project 1) Collect 30 anesthetized flies in eppendorf tube and freeze at -80°. Buffer A 100 mM Tris-HCl, pH 7.5 LiCl/KAc Solution II. Digestions (Sau3A I, HinP1 I, or Msp I--done separately) Set up 2 separate digestions:
1) Incubate 2.5hrs at 37°. III. Ligations
1) Incubate O/N at 4°. IV. PCR Set up PCR reactions with primers appropriate for the type of P element and the end of the element from which you want to recover genomic sequence (see table below and Figures).
Primers for PCR:
The Pry2/Pry1 combination has a higher annealing temperature than the Pry4/Pry1 and Pry4/Plw3-1 combinations, but the resulting PCR products do not allow sequencing directly off the 3' end of the P-element. The latter primer combinations are therefore used in all initial experiments; the Pry2/Pry1 combination can be used in those cases where strong and unique bands do not result. Cycle on MJ Research Thermal Cycler using the following parameters (~3hr run): 1X: 95° 5 min. Note that these parameters have been optimized for the MJ Research cycler; other thermal cyclers have different cycling profiles and adjustments may be necessary to achieve optimal results. Examine 5 ul of the PCR reactions on 1.5% agarose gel.
PCR Primer Sequences (5' to 3')--also see Figures: V. Sequencing Generally strong and unique bands result from the above PCR conditions, which can be directly sequenced without extensive purifications. Prior to sequencing, simply pass PCR reactions through Qiagen PCR purification columns (P/N 28104) to remove primers and nucleotides and elute in 50-80 ul of 10 mM Tris. This step is essential when dye terminator or standard S-35 chemistry is used for sequencing and helpful when dye primer chemistry is used; although in this latter case it may not be essential when the primers used for sequencing are internal to those used for PCR. Cycle Sequencing Protocol Cycle sequence using protocols appropriate for your sequencing method. The following cycle sequencing protocol is used by Garson Tsang of BDGP (garson@fruitfly.berkeley.edu). Currently, we are using ABI 377XL Automated Sequencers, FMC Bioproducts Long Ranger gel solution (P/N 50611), and the Perkin Elmer ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit (P/N 4303154). The use of other thermal cyclers and sequencing strategies will require different cycling parameters and sequencing protocols. 1) Mix the following in 200 ul PCR tubes: ABI BigDye Mix 3.0 ul PCR Template 2.5 ul Primer (1 uM) 1.5 ul 5X ABI dilution buffer 1.5 ul ddH2O 6.5 ul2) Cycle Sequence in a Perkin-Elmer 9700 (~2.5 hours) 96° 10 sec./ 50° 5 sec./ 60° 4 min. cycle 25 times followed by 4° hold 3) To purify reactions add cold 70% EtOH, let stand 20 minutes at RT, centrifuge for 30 minutes, discard EtOH and dry 4) Resuspend in 2.5 ul of the following: 5 parts (Deionized formamide) to 1 part (25 mM EDTA; 50 mg/ml Blue Dextran) 5) Load 1.9 ul onto gel Pharmacia ALF Sequencers have also been used for direct cycle sequencing of PCR products using dye primer technology. Good results were obtained using the inexpensive Silver Sequencing System (Promega P/N Q4131 - for DNA Sequencing reagents only). It should be noted that the buffer and dideoxy/deoxy nucleotide mixes that come with this kit are not proprietary and can be easily prepared from stock reagents. The ALF sequencers are apparently more sensitive to excessively strong signals therefore the amount of PCR product added to the cycle sequencing reactions may need to be adjusted, especially in the case of very small products, in order to obtain acceptable sequence reads. Standard S-35 sequencing should work as well; however, cycle sequencing protocols should be used for the sequencing reactions. Sequencing primers--also see Figures. The following primer sets are designed to sequence both ends of PCR products recovered from PlacW and PZ strains:
The PCR products recovered from PEP strains are sequenced with only one primer:
Primer Sequences (5' to 3')---also see Figures:Melting temperatures of sequencing primers:
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