Dec 13, 2024
IDT Primer and gBlock Hydration and Aliquots SOP
- Justine C. Condon1,
- Lisa M. Durso1
- 1USDA-ARS
Protocol Citation: Justine C. Condon, Lisa M. Durso 2024. IDT Primer and gBlock Hydration and Aliquots SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1r77lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 25, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 108738
Keywords: IDT Primers, DNA oligos, Probes, gBlock, Rehydrating, Primers, resuspension
Funders Acknowledgements:
USDA-ARS
Grant ID: NP212 Soil and Air
Disclaimer
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.
Abstract
This protocol describes the correct procedures on how to order primers, probes, and gBlocks; how to prepare them to a 100μM concentration; and how to aliquot them for future PCR applications.
Image Attribution
Stock Photo
Guidelines
Scope:
Primers are short, single strands of DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid), typically ranging from 18-22 bases that are used to initiate DNA synthesis. Primers are used because of their temperature stability in applications such as DNA sequencing and polymerase chain reaction (PCR). Probes are small sequences of DNA or RNA labeled with a reporter molecule (typically a fluorescent dye) used to detect the presence of a very specific DNA or RNA fragment.
PCR is a technique that enables researchers to rapidly and efficiently amplify (copy) a specific region of DNA or RNA into millions of copies. Primers are designed to be complementary to the beginning and end of the target sequence that will be amplified. In a PCR, it is the primers that dictate exactly what sequence of DNA gets copied. Primers are designed as pairs (forward and reverse) and enable DNA polymerase to attach and extend the new sequence. Probes, in conjunction with primers, are used for the detection of specific fragments in quantitative PCR (qPCR) reactions. Primers and probes can be designed using software and/or using known DNA sequences.
gBlocks Gene Fragments are double-stranded DNA fragments between 125-3000bp in length, designed for easy gene construction or modification. The gBlocks contain the primers (forward and reverse complement) of the gene of interest and serve as a synthetic positive control. gBlock sequences can either be obtained from literature research articles, or the NCBI (National Center for Biotechnology Information) nucleotide database.
IDT website: Integrated DNA Technologies | IDT
Responsibility:
This SOP applies to all staff members and students. These individuals must be knowledgeable about the requirements set forth within this document. The lab manager or designee shall ensure that all staff and students know the proper techniques.
Disclaimer:
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.
Appendix C - Calculations
To make a 10μM dilution:
V1*C1 = V2*C2
V1*100μM = 30uL*10μM
V1*100μM = 300uL*μM
V1=300μL*μM / 100μM
V1 = 3μL
Example: Make 30μL of the 10μM stock
27μL of molecular grade water
+ 3μL of the 100μM stock
30μL of the 10μM working stock
Amount Delivered to reach concentration of 10ng/μL:
V1*C1 = V2*C2
1μL * 500ng = V2 * 10ng/μL
500ng*μL/10ng = V2
50μL = V2
Manual Calculations:
**New calculations will need to be done every time a gBlock is ordered**
| A | B | |
| gBlock | # of base pairs | |
| GB3-sul1 | 344 bp | |
| GB4-intI1 | 494 bp | |
| GB5-erm(B) | 370 bp | |
| GB6-ctxm32 | 296 bp | |
| GB7 -16S, sul1, intI1 | 438 bp | |
| GB8-bla-ctxm1, ermB | 461 bp |
1. To calculate copy number / μL of the gBlock stock tube
(C)(M)(1 x 10-15 mol/fmol)(6.023 x 1023) = copy number/μL
**C & M will come from the Properties section of the IDT form**
C: current concentration of the gBlock Gene Fragment in ng/μL
(Should be the 10ng/μL from the newly hydrated gBlock)
M: molecular weight in fmol/ng
--Example: GB3-sul (from gBlock Specification Sheet - Appendix A, Step 44)
(10 ng/μL)(4.71 fmol/ng)(1 x 10-15 mol/fmol)(6.023 x 1023 copies/mol) = 2.835 x 1010 copies/μL
(1 ng/μL)(4.71 fmol/ng)(1 x 10-15 mol/fmol)(6.023 x 1023 copies/mol) = 2.835 x 109 copies/μL
2. To calculate concentration of gBlock to make 108 copies/5μL
C1V1 = C2V2
2.835 x 109 copies/μL x V1 = 5000 μL x (108/5µL) = 35.27 μL
3. Add 35.27 μL of gBlock stock to 4,964.73 μL TE buffer to create 5mL of 108 copies/5μL. Wet the pipette tip 5x before pipetting.
4. Wet pipette tip 5x, then aliquot the 108 copies/5μL standard into five 1mL tubes to keep as working stock to make serial dilutions. Clearly label and date.
- Can further aliquot 155μL into 0.2mL PCR strip tubes to prevent continual freeze/thaw
Materials
- Aerosol barrier no retention pipette tips (various volumes 1-1000µl)
- Pipettes for various volumes from 1-1000µl
- Microcentrifuge (Heraus Pico 21 Centrifuge, Thermo-Scientific, Waltham, MA)
- Fine-tip Sharpie Markers (black, blue, red ink)
- Cryo round labels (T-SPOTS, Microtube Tough-Spots, Dedham, MA)
- Vortex (Vortex Genie, Scientific Industries, Bohemia, NY)
- Molecular Biology Grade WaterHyCloneCatalog #SH30538.01
- Tris-EDTA buffer solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #93283-100ML
- 8-strip 0.2mL PCR tubes (T320-2N, Simport Scientific, Quebec, Canada)
- CRYOELITE 2MLAndwin ScientificCatalog #02-912-729
- Sterile Nitrile Examination Gloves
- Primers and Probes (Integrated DNA Technology, Coralville, IA)
- gBlocks (Integrated DNA Technology, Coralville, IA)
- Freezer boxes (71001-642, Wilson Dependable Services, Washington D.C.)
- -20°C Freezer
- 4°C Refrigerator
- 50°C Incubator (or dry/water bath)
Safety warnings
Wear proper personal protective equipment (PPE) at all times when handling DNA: sterile nitrile gloves and lab coat.
gBlocks must be hydrated in another space from where you set-up PCR reactions.
Before start
gBlocks must be hydrated in another space from where PCR reactions are set-up.
Procedure - Ordering Primers
Procedure - Ordering Primers
In the menu under ‘Products and Services’ > ‘DNA & RNA’ select ‘Custom DNA Oligos’ > ‘DNA Oligos.’ Then ‘Order Now.’
↓
Insert the information of the primers of interest (name, scale, sequence 5’-3’, formulation, purification, etc. (Name: include primer lab ID #, gene name, and ‘Fwd’ or ‘Rvs’),
Example: LDP1-16S-Fwd, 100 nmol DNA oligo, CCTACGGGAGGCAGCAG, No Formulation, Standard Desalting.
Note
When inputting the sequence, be mindful not to have gaps or spaces between the nucleotide bases.
When ready, ‘Check out.’ The order should arrive in the mail within a few days.
Procedure – Ordering Probes
Procedure – Ordering Probes
Search for ‘PrimeTime Probes’.
The current lab probes use the FAM reporter dye and ZEN/Iowa Black Quencher.
Input the ordering information: Name of probe (include primer lab ID #, gene name, and ‘P’ for probe), scale, type of dye/quencher, sequence.
Example: 16S: Name - P28-16S-P ; Scale – 100nmole ; Dye/Quencher - 5’ 6-FAM/ZEN/3’IBFQ ; Sequence - CTTGTACACACCGCCCGTC
Note
When inputting the sequence, be mindful not to have gaps or spaces between the nucleotide bases.
Check inventory first, but if low or out, order additional PrimeTime Gene Expression Master Mix (25mL, cat # 1055771). The PrimeTime mix contains a hot-start antibody, Taq polymerase, and other components needed for probe-based qPCR
When ready, ‘Check out.’ The order should arrive in the mail within a few days.
Procedure – Ordering gBlocks
Procedure – Ordering gBlocks
Search for ‘gBlocks Gene Fragments,’ then ‘Order Tubes.’
Fill in the required name and sequence information. Then test the complexity of the sequence. This will let you know if there are any problems with the sequence and any suggestions on how to improve it.
Note
When inputting the sequence, be mindful not to have gaps or spaces between the nucleotide bases.
When ready, ‘Check out.’ The order should arrive in the mail within a few days.
Procedure – Preparation and Aliquots – Primers and Probes
Procedure – Preparation and Aliquots – Primers and Probes
21m
21m
Note
Precise pipetting is important for all transfers.
Safety information
At all stages of this process, wear Sterile Nitrile Examination Gloves to avoid potential contamination.
Color Coding: Forward primer = Blue, Reverse Primer = Black, Probe = Red
When IDT packages arrive in the mail open the package containing the dry, lyophilized product and store at -20 °C until they need to be prepared for any project that requires them.
- With a sharpie, label the top of each tube with its ID, and write the received date and your initials on the side of each tube.
Before starting any DNA work for a project, remove the primers (or probe) from the freezer (-20 °C ).
Centrifuge the tubes to get the dry flakes to the bottom.
Using the specification sheet (Appendix A go to step #44 ), look for the “Amount of Oligo” section (outlined in the red box).
Each specification sheet will indicate a different amount of molecular biology grade water that will be needed to produce primers (or probe) at a 100 micromolar (µM) concentration.
Pipette the amount needed to produce the 100 micromolar (µM) primers (or probe).
- Using a round, colored cryo label, label the top of each tube with the ID (ie P28-16S-Fwd) and color coding listed above in go to step #16 . This will be an indication that the tube has been hydrated.
Vortex the primers (or probe) for 00:00:30 , and centrifuge briefly.
30s
Let the primers (or probe) hydrate for at least 00:20:00 (or Overnight ) in the refrigerator.
- The hydrated tube will be the Stock tube.
- Dilutions and aliquots will be the Working Stock.
20m
When ready to use, vortex the Stock Tube (00:00:30 ) and centrifuge again before use.
- The 4GU, 4GM, Tet, and invA assays use the 100 micromolar (µM) concentration, so proceed to go to step #27 for aliquots.
30s
The RD Probe-based qPCR uses 10µM concentrations. See [Appendix C in the Guidelines tab] for the calculation steps and equations to make 30µl aliquots, then follow go to step #27 -go to step #28 .
Label a PCR tube with a recognizable and unique identifier. Date the side of the tube. (See color coding in go to step #16 ).
Aliquot 30 µL of primer (or probe) into each PCR tube and prepare 16 tubes worth of aliquots at a time.
Seal the PCR tubes containing the primer (or probe) aliquots.
For Sample DNA Product: To make a working stock dilution of sample DNA that delivers at 1µl/rxn at a 5 µL volume: create a dilution at the desired volume in increments of 1 µL DNA + 4 µL PCR water.
Example: for 25 µL of working stock sample DNA, mix 5 µL DNA with 20 µL PCR water.
Place the aliquots into a labeled freezer box.
Note
Note: The labeling used for the freezer box should be specific to a given project or genes of interest.
Store the labeled freezer box of primer, probe, or sample aliquots at-20 °C .
Anytime a set of primers (or probe) is needed for an experiment, just pull one tube of required Forward and Reverse primers and/or probe from the freezer and thaw for use. Keep in a cold-block to keep cool while using.
Note
Use a razor to cut between the tubes – DO NOT tear the tube off – can cause breaks in the tube, or adjacent tube, leaving an open hole and leading to evaporation.
Discard any unused primer (or probe) from the pulled and thawed aliquot to avoid contamination of the reserve.
Procedure – Preparation and aliquots – gBlocks
Procedure – Preparation and aliquots – gBlocks
8h 20m 5s
8h 20m 5s
Safety information
gBlocks MUST BE HYDRATED IN A SEPARATE LAB to avoid contamination.
gBlocks must be hydrated in another space from where you set-up PCR reactions.
Note
Precise pipetting is important for all transfers.
Safety information
At all stages of this process, wear Sterile Nitrile Examination Gloves to avoid potential contamination.
When IDT packages arrive in the mail open the package containing the dry, lyophilized gBlock and store at-20 °C until they need to be prepared for any project that requires them.
- With a sharpie, label the top of each tube with its ID, and write the received date and your initials on the side of each tube.
Before starting any DNA work for a project, remove the gBlocks from the freezer (-20 °C ).
Centrifuge the tubes for 00:00:03 -00:00:05 at a minimum of 3000 x g to ensure the material is at the bottom of the tube.
5s
gBlocks need to be at a concentration of 10ng/µL.
- Using the specification sheet (See Appendix A go to step #45 ), look for the “Amount Delivered” in the ‘Properties’ section (outlined in the red box) stating how much dsDNA is in the tube.
- In the specification sheet example the starting amount is 500 ng , 50 µL will be added to get 10 ng/µL (See Appendix C for the steps and equations in the Guidelines tab).
Wet the pipette tip 5x, and add Tris-EDTA (TE) to reach a final concentration of 10 ng/µL .
Vortex the tube, and incubate at 50 °C for 00:20:00 (can use a dry bath or water bath).
20m
Briefly vortex again, and centrifuge. Leave Overnight at 4 °C before aliquoting (See Appendix B go to step #46 for diluting to copy number and standards).
- The hydrated tube will be the Stock tube.
- Dilutions and aliquots will be the Working Stock.
8h
Appendix A – Specification Sheet Examples
Appendix A – Specification Sheet Examples
Primer Specification Sheet
gBlock Specification Sheet
Appendix B - gBlock dilute to copy number (101-108 copies)
Appendix B - gBlock dilute to copy number (101-108 copies)
15m 5s
15m 5s
Serial Dilutions for standard curve
Label 0.2mL strip tubes (108 to 101) and UV for 00:15:00 .
15m
Wet the pipette tip, and transfer 45 µL of TE into the tubes labeled 107 – 101 (this can be done in the biosafety hood), then spin briefly
Using the freezer stock of the 108 copies/5µl gBlock standard created.
- Wet pipette tip 5x, and transfer 50 µL of the 108 copies/5µl standard into the first tube labeled 108.
- Continuing with the first strip tube, wet pipette tip 5x, and transfer 5 µL into the 107 tube. Then rinse the tip in the diluent 5x to expel all DNA. Vortex for 00:00:05 , spin briefly, and repeat steps for all remaining tubes to the 101. (Do not push on the bottom of the tube with pipette tip).
5s
Keep fresh at 4 °C for one week, or freeze immediately for longer storage.
Protocol references
Dungan, R.S., C.W. McKinney, A.B. Leytem. 2018. Tracking antibiotic resistance
genes in soil irrigated with dairy wastewater. Science of the Total Environment 635”1477-1483.
IDT website: Integrated DNA Technologies | IDT