Aug 01, 2024

Public workspaceHome made SM 100bp DNA ladder for agarose gel

DOI
dx.doi.org/10.17504/protocols.io.dm6gpz535lzp/v1
  • 1LIttoral ENvironement et Sociétés - UMR 7266 - CNRS - La Rochelle Université
Stéphane Mauger
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpz535lzp/v1
Protocol Citation: Stéphane Mauger 2024. Home made SM 100bp DNA ladder for agarose gel. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz535lzp/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: August 01, 2024
Last Modified: August 01, 2024
Protocol Integer ID: 104413
Keywords: double-stranded DNA sizing, agarose gel, 100pb ladder
Abstract
The SM 100pb DNA Ladder is a standard size marker equivalent to the Fisher’s O'RangeRuler‱ 100 pb DNA Ladder (#SM0623). The SM 100pb Ladder allows to determine the size of double-stranded DNA fragments between 100 bp and 1500 bp and it is composed of 15 double-stranded DNA fragments of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 and 1500 bp. Like the O'RangeRuler 100 bp, the SM 100 bp gives more intense bands at 500, 1000 and 1500 bp.

At the end, the SM 100 pb DNA Ladder is 25 times cheaper than the O'RangeRuler 100 pb.

Map of the universal cloning vector pGEM‱-3Z(+) showing the position of the 16 primers


Comparaison between O'rangeRuler 100pb (Thermofisher) and SM100bp ladder on 2% agarose gel


Protocol materials
ReagentGoTaq® Flexi DNA PolymerasePromegaCatalog #M8291
ReagentpGEM®-3Zf( ) VectorsPromegaCatalog #P2271
Primers and pGEM®-3Zf(+) Vectors preparation
Primers and pGEM®-3Zf(+) Vectors preparation
Oligo primers dilution
in 16 Amount1.5 mL microtubes, dilute 1:10 each oligo primer at 100µM to final concentration 10µM, see the SM100pb_primers.xlsx file below.
Amount50 µL oligo primer (100µM)
Amount450 µL nuclease free water

Homogenize and store at Temperature4 °C (or at Temperature-20 °C for a long-term storage).

SM100pb oligo primers sequences (salt free purification):

Download SM100pb_primers.xlsxSM100pb_primers.xlsx12KB

pGEM®-3Zf(+) Vectors dilution
In Amount1.5 mL microtube, dilute 1:100 pGEM®-3Zf(+) vector at 1µg/µL to final concentration 10ng/µL.
Amount5 µL pGEM®-3Zf(+) (1µg/µL)
Amount495 µL nuclease free water

Homogenize and store at Temperature4 °C (or at Temperature-20 °C for a long-term storage).

ReagentpGEM®-3Zf(+) VectorsPromegaCatalog #P2271

PCR amplification to generate 100pb-1500pb double-stranded DNA fragments
PCR amplification to generate 100pb-1500pb double-stranded DNA fragments
10s
10s
PCR mix preparation
SM100pb is produced using 28 PCRs amplifications in a total of Amount50 µL reaction volume:
1 x Amount50 µL for 600pb, 700pb, 800pb, 900pb, 1100pb, 1200pb, 1300pb and 1400pb fragments
2 x Amount50 µL for 200pb, 300pb and 400pb fragments
3 x Amount50 µL for 1000pb and 1500pb fragments
4 x Amount50 µL for 100pb and 500pb fragments

Defreeze and vortex all reagents, except enzymes (stored at Temperature-20 °C ), for approximatelyDuration00:00:05
Spin down all reagents for approximately Duration00:00:05 and place TemperatureOn ice .

In Amount1.5 µL microtube, prepare the PCR mix according to the following table :
ABCDE
Initial concentrationFinal concentrationn=1n=28
SMF forward primer10µM400mM2µL56µL
Green GoTaq buffer5X1X10µL280µL
MgCl225mM1mM2µL56µL
pGEM®-3Zf(+) vecto10ng/µL20ng2µL56µL
dNTP mix2.5mM150µM each3µL84µL
GoTaq polymerase5 u/µL1.75U0.35µL9.8µL
nuclease free water28.65µL802.2µL
TOTAL48µL1344µL
PCR mix composition

ReagentGoTaq® Flexi DNA PolymerasePromegaCatalog #M8291

10s
Reverse primers and mix combinaison
Defreeze and vortex all the 15 reverse oligo primers at 10µM
In a 96-well plate PCR, transfer Amount2 µL of each reverse oligo primers (10µM) according to the following map :
ABCDEFGH
600R200R1000R500R
700R200R1500R500R
800R300R1500R500R
900R300R1500R500R
1100R400R100R
1200R400R100R
1300R1000R100R
1400R1000R100R
Map of the PCR plate

Vortex and spin down the PCR mix tube, transfer Amount48 µL in each of the 28 wells.
Seal the PCR plate.

In thermocycler, run PCR amplification with cycles follows:
ABCD
Cycles stepTemperatureTimeCycles
Initial denaturation94°C5 min1
Denaturation94°C30 sec40
Annealing60°C30 sec40
Extension72°C30 sec40
Final extension72°C60 min1
Hold4°C
PCR program

After PCR, pool and dilute 1:2 the PCR amplification
In a Amount5 mL tube:
Amount1400 mL of PCR amplification (28 x Amount50 µL )
Amount420 µL 5X green GoTag buffer
Amount980 µL nuclease free water

Homogenize and store at Temperature4 °C (or at Temperature-20 °C for a long-term storage).

Load Amount5 µL to Amount10 µL per line in a agarose gel.

Comparaison between O'rangeRuler 100pb (Thermofisher)
and SM100bp ladder on 2% agarose gel