|Title||Digoxigenin-labelled Riboprobe Synthesis from a PCR-generated DNA template|
Digoxigenin-labelled Riboprobe Synthesis from a PCR-generated DNA template by the Little Group
Primers are designed to amplify a 3’UTR region of a gene in the FANTOM2 pool of between 500-700bp. The reverse primer is linked to a T7 polymerase promoter tag sequence as follows:
Reverse (3’) Primer 5’ leader sequence CGATGT- T7 polymerase sequence TAATACGACTCACTATAGGG – then the primer sequence – 3’.
Primers are ordered from Invitrogen.
Re-suspend PCR primers using 10mM Tris-HCl pH8.0 to make a 100µM stock. From the well resuspended stock solution, make a 20µM working dilution. At this point, you may combine both the forward and reverse primer for each set into a labeled 1.5ml eppendorf.
Eg. Dilute stock ie. 10µl F primer stock + 10µl Reverse primer stock + 30ul 10mM Tris pH8.0 in a 1.5ml eppendorf.
Final concentration is 20µM = 20pmol/ul.
Store stocks at -20°C
To determine the optimal annealing temperature, choose 3-5 primer sets and perform a gradient PCR as follows using the BioRAD MyCycler. One reaction volume is to be divided equally between 8-strip 0.2ml PCR tubes (Scientific Specialties Inc. 3240-00):
PCR Conditions using BioRad MyCycler
* The gradient depends on melting temperature of primer set and spans across 8 temperature points in the PCR machine.
Run 12.5ul on a 1% agarose gel at 100V for 35min to determine the optimal annealing temp.
Once the optimal annealing temperature has been determined, prepare a primary PCR reaction mix for the 48 primer sets in a 96well plate as follows:
Make enough Master Mix to include negative controls for the PCR.
Use every second column of the 96 well plate.
Cover with sealing film.
Spin at 1500rpm for 5min.
Perform PCR in the Tetrad DNA Engine (Microarray Facility) as follows:
* Annealing temp based on results of gradient PCR
Run out 50uL product on 1.5% agarose gel 100V for 25min. Expect to see 1 distinct band between 500-700bp.
Cut bands out using a scalpel blade under UV light and placed into appropriately numbered 1.5ml tubes.
Purify PCR products using Qiagen Gel Extraction Kit as summarised:
Store samples @ 4°C or -20°C for long term storage.
If no primary PCR product is amplified, repeat reaction using E15.5 whole embryo cDNA instead of the FANTOM pool.
Record the percentage of primary PCR which failed to amplify with the FANTOM pool.
Also record if the product failed to amplify using the cDNA.
Perform secondary PCR on purified primary PCR template. For one reaction:
PCR conditions (using Tetrad in Array facility or DNA Engine in MHL Lab)
After completion of secondary PCR run 5uL of sample on 1.5% agarose gel 100V 25min to check amplification and concentration.
Purify products using Qiagen PCR Purification Kit as summarised:
Store sample @ 4°C
The DNA concentration is measured using 2µl on a Nanodrop.
If the concentration is <40ng/µl, the sample will be concentrated using a Speed Vacuum.
Record the percentage of secondary PCRs which failed to amplify.
4 primer sets (A1/2.A3/4, D1/2, H11/12) are chosen and secondary PCR products are sequenced checked.
Use 5-20ng DNA template (500-1000bp).
Cycling Conditions using DNA Engine
| 1. | 96°C | 1min | 1 cycle |
Post-reaction Clean Up
Use the magnesium sulphate protocol for the clean-up of sequencing reactions as provided by AGRF.
Once the sequencing samples are returned:
in vitro Transcription of Digoxigenin-Labeled Riboprobes
Once sequences and orientation are confirmed, continue with the production of riboprobes.
Ensure RNase-free conditions are maintained and reagents kept on ice.
Incubate in heating block @37°C for 60min.
Add another 1uL T7 RNA polymerase.
Incubate in heating block @37°C for 60min
Add 2.0uL RQ1-DNaseI (10U/uL) @ 37°C for 20 min.
Purify using Roche Quick Mini Spin Column:
Prepare Roche column - flick & invert to resuspend matrix.
Snap off appropriate plastic at top and bottom of column.
Spin column @ 3700 rpm for 1min to remove buffer (collected in 2.0mL tube)
Load riboprobe onto centre of column placed in 1.5ml eppendorf labelled with appropriate sticky label ‘Gene Symbol-Riboprobe’
Spin @ 3500rpm for 4min. Collect approximately 50uL each tube.
Run out 2uL of probe on 1.5% agarose gel at 100V for 15min
Check product on UV Transilluminator. Ideal result shows distinct bright bands with no smearing.
Print 2 pictures of gel and save in GUDMAP folder with date
Store at -70°C in GUDMAP Riboprobe Boxes
Record the percentage of riboprobes which failed to be transcribed.
Quantify riboprobe using 2µl sample on the Nanodrop.
The concentration is usually between 50-300ng/ul.
Record concentration on excel spreadsheet and tube.
|Digoxigenin-labelled Riboprobe Synthesis from a PCR-generated DNA template.pdf|
|Release Date||2017-08-04 12:22:29|
|Principal Investigator||Melissa H. Little, MCRI|
|Murdoch Children’s Research Institute|