Long ssDNA Gel Extraction Kit for 3kb

Product#: DS640
$300.00
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Long ssDNA Gel Extraction Kit for 3kb

Cat. No. DS640
Store  Stable at 15 to 25℃ for 24 months from the date of receipt
Size 25 tests
Caution! Wear gloves and protective clothing while handling the Crystal Violet

Description

The Long ssDNA Gel Extraction Kit is a highly-specialized kit for long ssDNA purification from agarose gel. The spin column, buffer formulations and purification protocol are optimized for high recovery yields and high purity of long ssDNA. Long ssDNA purified with the kit is high-quality, with low mechanical degradation and without ultraviolet light-induced damage from the purification process. The purified long ssDNA is suitable for molecular biology and biotechnology applications. 

The Long ssDNA Preparation Kit can be universally used for purification and clean-up of long ssDNA.

Kit Components: 25 reactions
  • Ethanol and isopropanol are required for the product.
  • If precipitated material has formed in Gel-Dissolving Buffer, heat to dissolve at 37℃.
  • Heat block are required for 50-70℃ incubation.
  • Denaturing Gel-Loading Buffer is required for the agarose gel electrophoresis of long ssDNA. It is not included in the kit. It can be purchased individually from BioDynamics Laboratory Inc. (see “Related Products“ on page 4).

Components

Contents

Size

Crystal Violet Solution

Crystal Violet 4mg/ml (2,500 x)

5 ml

Gel-Dissolving Buffer

It contains guanidine thiocyanate as chaotropic salts

45 ml

Wash Buffer 1

Tris-base buffer (Add 45 ml of 100 % ethanol to the Wash Buffer before use.)

8 ml

Wash Buffer 2

Tris-base buffer (Add 45 ml of 100 % ethanol to the Wash Buffer before use.)

11 ml

Elution Buffer

10 mM Tris-HCl, pH 8.0.

5 ml

Spin Column

 

25 pieces

Collection Tube

 

25 pieces


Features and Specifications: 
  • Optimized for long ssDNA.
  • High recovery yield (typically 75-90%).
  • High purity.
  • Low mechanical degradation.
  • No UV light-induced DNA damage.
  • Direct monitoring of the migration of long ssDNA blue bands in the gel during electrophoresis.
  • Excision of long ssDNA under ambient light.
  • Guanidine thiocyanate is used as chaotropic salts. NaI is not used.*1
  • ssDNA size for excision: 500 - 3,000 bases. *2
  • Binding capacity on a Spin Column for ssDNA binding is up to 5μg.
  • Elution volume: ≥ 15 μl.
*1 Residual NaI may be difficult to remove, and reduces the efficiency of downstream enzymatic reactions.
*2 200 bases of long ssDNA also can be extracted using the kit, but the recovery yield is not so high (about 40-45%).

Protocol of DNA excision and purification:
STEP 1: Preparation of agarose gel containing Crystal Violet
An example of this is given for making 100 ml of a 1.2% agarose gel.
  • Add 1.2 g of agarose powder to 100 ml of 1×TAE buffer and mix them.
  • Dissolve the agarose in the microwave.
  • Add 40μl of Crystal Violet Solution to the agarose (100 ml) and mix them by swirling.
  • Pour the agarose into a mold and set a comb.* Allow the gel to harden.
* It is recommended to make thick gel with deep wells so as not to cause the overflow of a loaded sample in a well,
resulting in contamination.

STEP 2: Agarose gel electrophoresis and excision
A simple way is shown in the following example.
  • Pre-chill 1xTAE running buffer containing Crystal Violet (add 40μl Crystal Violet /100ml 1xTAE) on ice or in a refrigerator. *1
  • Put the horizontal gel electrophoresis apparatus on ice in an ice bucket in a draft chamber. *2, *3, *4
  • Pour pre-chilled 1xTAE running buffer containing Crystal Violet into the electrophoresis apparatus. *5
  • Put the agarose gel into the electrophoresis apparatus.
  • Prepare loading samples by mixing the long ssDNAs sample (e.g. the nicked plasmid) and 3 volumes of the Denaturing Gel-Loading Buffer. *6, *7, *8
  • Heat the mixture at 70°C for 5 min, then chill on ice for 1 min. Subject the mixture to agarose gel electrophoresis. *9, *10
  • Run the gel at 100 V at a low temperature (less than 20℃). *2, *3, *4
  • Monitor the long ssDNA blue bands moving in the gel during electrophoresis (Figure 1). Stop the electrophoresis after the bands are sufficiently resolved. *11, *12
  • Excise the long ssDNA band of interest from the agarose gel with a scalpel.*13, *14
 

DS640_Fig1.jpg





Figure 1. Agarose gel electrophoresis using the gel containing Crystal Violet.
1.2% agarose gel in 1x TAE, 5.5cm x 6.0 cm. The loading volume of each sample is 24 μl.

Lane 1: pLSODN-1 (1.5 Kb Fragment), double-nicked and denatured *15 12 μg
Lane 2: pLSODN-3 (3 Kb Fragment), double-nicked and denatured *15 12 μg


STEP 3: DNA extraction and purification
Before start:
  • Add 45 ml of 100 % ethanol to Wash Buffer 1 and Wash Buffer 2 bottles, respectively.
  • Isopropanol is required in the step.
  • All centrifugation steps should be carried out at 16,000×g (around 13,000 rpm in a conventional microcentrifuge) at room temperature (20℃-25℃). Centrifugation at lower temperature might affect long ssDNA yield.
  • This kit can also be used for long ssDNA clean-up from enzymatic reactions. For this purpose, add 3 volumes of Gel-Dissolving Buffer and 1 volume of isopropanol to the reaction, mix well, and proceed with step 5 of the protocol.
  • Transfer the gel slice to a 1.5 ml microcentrifuge tube and weight the gel slice.
  •  Add 3 volumes of Gel-Dissolving Buffer.
  • Incubate the tube at 50℃, vortexing periodically until the gel slice is completely dissolved for 10-15 min. *1
  • Add one gel volume of isopropanol to the dissolved gel and mix well.
  • Insert a Spin Column into a Collection Tube.
  • Load the sample to the Spin Column and centrifuge for 1 min. Discard the flow-through in the Collection Tube with a 1ml pipette tip. *2, *3, *4
  • Centrifuge the Spin Column again for 1 min. Remove the residual flow-through completely with a 10 μl or a 100 μl pipette tip. *5
  • Add 500 μl of Wash Buffer 1 to the Spin Column and centrifuge for 1 min. Discard the flow-through in the Collection Tube with a 1ml pipette tip (1st Wash). *3, *4
  • Repeat wash with Wash Buffer 1 (Step 8, 2nd Wash). *3, *4
  • Add 500 μl of Wash Buffer 1 to the Spin Column, close the cap tightly and vortex for 5 seconds to wash the whole inner wall of the Spin Column (3rd Wash).
  • Centrifuge for 1 min. Keep the flow-through in the Collection Tube and vortex for 5 seconds to wash both the outer wall of the Spin Column and the inner wall of the Collection Tube.
  • Centrifuge for 1 min. Discard the flow-through in the Collection Tube using a 1ml pipette tip. *3, *4
  • Add 500 μl of Wash Buffer 2 to the Spin Column and centrifuge for 1 min. Discard the flow-through in the Collection Tube with 1ml pipette tip (4th Wash). *3, *4
  • Centrifuge the Spin Column again for 1 min to remove residual Wash Buffer 2 completely.
  • Transfer the Spin Column into a new microcentrifuge tube.
  • Add 15-40 μl of Elution Buffer onto the Spin Column and incubate it at 70℃ for 5 min. *6
  • Directly Centrifuge the Spin Column for 1 min to elute long ssDNA. *7 

DS640_Fig2.jpg


Figure 2. Agarose gel electrophoresis of purified long ssDNAs.

1.2% agarose gel in 1x TAE. 5.5cm x 6.0 cm.




Lane 1: pLSODN-1 (1.5 kb Fragment), double-nicked and denatured 800 ng

Lane 2: a long ssDNA purified from pLSODN-1 (1.5 kb Fragment) 400 ng

Lane 3: pLSODN-3 (3 kb Fragment), double-nicked and denatured 800 ng

Lane 4: a long ssDNA purified from pLSODN-3 (3 kb Fragment) 400 ng


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