Important considerations
2.3 SOURCING AND DEVELOPING MICROSATELLITE LOCI
2.3.4 DNA Sequencing
Where possible all loci were sequenced for the blue crane at loci obtained from two microsatellite sources:
1. Existing microsatellite loci previously developed for other crane species (section 2.3.1 and 2.3.2) that successfully amplified cross-species in the blue crane. These loci were sequenced to check for the presence of a microsatellite repeat, thereby confirming amplification of a microsatellite locus required for this study. Sequences also allowed for the redesign of primers, where necessary, based on blue crane sequences.
2. All loci developed from the two blue crane microsatellite libraries (section 2.3.3). These loci were sequenced to allow primer design and tests for polymorphism.
All loci were cloned using a protocol based on 'Cloning of PCR products' by Sambrook et al. (2000) followed by a precipitation reaction to obtain clean DNA fragments for sequencing on an ABI 3730. Sequences were initially generated by Gavin Horsburgh at SMGF (University of Sheffield). Those loci for which sequences originally failed to be
generated were re-sequenced by myself. All blue crane sequences generated in this study are provided elsewhere (Appendix Ill).
An initial PCR was performed and the products run on a 2 % agarose gel to determine fragment size. This identified whether optimisation of the PCR conditions had been achieved. If a weak band was observed, the PCR reaction volume was increased to 20 ,.il, and the volume of PCR product added during ligation was increased to 6 Ill. Ligation using the PCR product can commence when a strong, single PCR product can be seen on an agarose gel, as this indicates the presence of a high number of fragments of the desired length.
When PCR products were non-specific and unsuitable for purification or sequencing due to the presence of multiple fragment sizes, these were re-cloned (by the author) following gel extraction of the correct fragment size. This was achieved by running the fragments on an agarose gel to separate the fragments according to size.The correct fragment size was cut out using a sterile blade for further purification using the 'QIAquick' gel extraction kit protocol (Qiagen). The cleaned fragment was then processed as before from the ligation step of the Sambrooket al. (2000) cloning protocol.
PCR purification can be performed using the gel extraction protocol provided with the QIAGEN gel extraction kit. This process will remove dNTPs and primers, a pre-requisite for ligation (step 3,below).
Cloning PCR products for sequencing
a) Ligation: once fragment has been gel extracted a ligation is set up using pGEM-T easy (Promega) as follows:
5 III2x Rapid ligation buffer 1~lpGEM-T easy (50 ng/ul) 3 ul PCR product
1 ul T4DNA Ligase (always keep on ice and add last)
Ligation is incubated overnight or weekend at 15DC in water bath kept in 4 DCroom.
b) Transformation:
1. For efficiency of transformation, electroporation was used to.enable transformants to be obtained with very weak products.
2. Add ddH20 to 500 ml then add 7.5 g agar. Autoclave.
3. Place the autoclave agar in microwave with lid loosened and cook until completely melted. Once melted,place in 55DCwater bath and allow to cool to that temp.
4. Add 0.5 ml of Ampicillin (50 mg/ml) and 0.6 ml of Extra Blue" plus (Qbiogene) (this is an alternative to IPTG/Xgal in Dimethyl Formamide) in 125 ml LB agar (recipe below).
LB agar (llitre)
5g Bacto™ Tryptone (l %) 2.5 g Bacto" Yeast extract (0.5%)
5g -NaCI (1%)
5. Pour plates suing aseptic techniques. A 500 ml yields 20+ plates (~25 ml/plate).
Two plates per ligation are needed. Dilute one in case too many transformants are obtained in the other. Once set,dry plates in 55DCincubator for 30-45 minutes.
c) Transformation by Electroporation:
1.Place the intended number of electroporation cuvettes in -20 DCfreezer.
2. LB is needed at this stage (made as above without agar added and autoclaved).
3.In 1.5ml Eppendorfs place 90 III ofLB for each transformation.
4. Place 3 f.!l of ligation into a separate Eppendorf with 2 f.!l ddH20, flick and spin down. Do this with allligations before the start (no need to keep on ice).
5. Add 40 f.!l of competent cells to ligation/ddll-O mix, flick to mix and spin down, place on ice(~1 min).
6. Into a chilled electroporation cuvette, place cell mix. Electroporator should be set to 2350 V. After the voltage has passed through the cuvette, remove the cuvette and immediately add 60 f.!l ofLB.Remove mix from cuvette into fresh 1.5 ml Eppendorf.
Take 10 /-Ll of mix and add to the Eppendorfcontaining 90ulofLB.Mix.
7. Take two agar plates
Cl
mark them N for neat and D for dilute) and add the transformation mix to both.8. Using aseptic techniques, spread culture as evenlyaspossible across agar surface.
Place Petri dishes agar surface up to allow culture to dry into agar.
9. Repeat for next ligation mix.
10. Place all plates agar surface down in 37 "C incubator. Incubate for~18 h.
11. In 96 well titre plates add 100 f.!l LB + 20 % glycerol. Using toothpicks, pick colonies into wells.
12. Incubate plates in 37 "C incubator with shaking at 110 rpm. Cover plates to prevent drying.
d) Sequencing
1. Perform peR as follows:
1 /-Ll 10xbuffer
1 III dNTPs
1III Ml3forward primer
1 ul M13 reverse primer 0.2~l MgCh
4.75 ul ddH20
0.05 ul Taq polymerase I ul bacterial culture Total 10 ul
PCR program:
95°C for 3 min; 30 cycles of95 "C for 1 min, 55°C for 1 min, 72°C for 1 min (can be extended to 2 min ifproduct larger); followed by 2 min extension at 72 "C.
2. Run 4 J..tl of product on appropriate percentage gel to determine if product present.
3. With rest of sample add 2 ~lEXO-sap and incubate on a PCR block for 15 min at 37°C then for 15 min at 80°C to inactivate enzymes.
4. Perform Sequence PCR onforward and reverse as follows:
1 ul Big Dye V1.1
1.5~l 5x sequencing dilution buffer 1.6~l Primer (M13F or M13R) 3 ul DNA (exo-sapped) 2.9 ul ddH20
Total 10 ul
Include a control as follows:
1 ul Big Dye V1.1
1.5~l 5x sequencing dilution buffer
2 ul M13 -21
2 ~l pGEM-3Zf(+)
3.5 ul ddH20 Total 10~l
Perform PCR as follows:
96°C for 1 min; 30 cycles of94 °C for 10 s, 50°C for 5 s, then 60°C for 4 min.
5. Precipitate reactions and sequence as using the protocol below based on Dawson et al. (2005a).
PrecipitationofBigDye Terminator vI.I cycle sequencing reactions
a.) Ethanol/EDTAlsodium acetate precipitation
1. Add 2 ul of 125 mM EDTA, 2 III 3M sodium acetate (pH 5.2), 10 III autoclaved ddH20 and 50III 100% ethanol to each tube.
2. Transfer 10 III of sequencing reactions to the labelled 1.5 ml eppendorf and briefly vortex.
3. Incubate at room temperature for 15 min in a dark drawer.
4. Spin at 13,000 rpm for 15 min. Orientate eppendorfs with the hinges outermost so the location ofthe DNA pelletis known.
5. Set a P200 pipette to 80 III and aspirate off the supematant, taking care not to dislodge the pellet.
6. Add 195 III70 % ethanol (pre-cooled at -70°C).
7. Spin at 13,000 rpm for 5 min
8. Repeat step 6 using a P200 pipette set to 200 Ill.
9. Air-dry at room temperature in the dark for 15 to 60 minutes or until dry.
b.) Sequencing on the ABI 3730 Analyser
1. Add 10 ul Formamide to wells and mix (pipette up and down with multi-channel)
2. Denature for 3 min at 95DC,place on ice until ready to load on the DNA analyser.
3. Sequencing was performed using the ABI 3730 DNA analyser and two sequencing PCR reactions were run for each sample. One reaction used the forward M13 primer, the second used the reverse M13 primer.
The final consensus sequences were created by comparing forward and reverse sequences.
Raw sequences were analysed and consensus sequences generated for all loci that amplified a PCR product in blue crane.