[EasyTools] DNA Dilution Calculator

DNA Dilution Calculator

Enter DNA concentrations (ug/ul) for up to 10 samples and specify the final volume (ul) to calculate dilution volumes.

Sample 1 (ug/ul):
Sample 2 (ug/ul):
Sample 3 (ug/ul):
Sample 4 (ug/ul):
Sample 5 (ug/ul):
Sample 6 (ug/ul):
Sample 7 (ug/ul):
Sample 8 (ug/ul):
Sample 9 (ug/ul):
Sample 10 (ug/ul):
Final Volume (ul):

Sample (ug/ul)	DNA (ul)	DW (ul)

Competent Cells Transformation: A Step-by-Step Guide

In this post, we present a concise overview of cell transformation protocols. Competent Cells transformation is a pivotal process in molecular biology and genetic engineering, and we'll provide essential information for successful experiments.

Competent Cells Transformation

: A Step-by-Step Guide

Here, we have summarized the protocols for One Shot® TOP10 Competent Cells and NEB® 5-alpha Competent E. coli (High Efficiency). Each protocol refers to the procedures included in the corresponding product.

One Shot® TOP10 Competent Cells

 

1. Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.

2. Thaw, on ice, one 50 μL vial of One Shot® cells for each ligation/transformation.

3. Pipet 1–5 μL of each ligation reaction directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at −20°C.

4. Incubate the vial(s) on ice for 30 minutes.

5. Incubate for exactly 30 seconds in the 42°C water bath. Do not mix or shake.

6. Remove vial(s) from the 42°C bath and place them on ice.

7. Add 250 μL of pre-warmed S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.

8. Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.

9. Spread 20–200 μL from each transformation vial on separate, labeled LB agar plates. The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.

10. Invert the plate(s) and incubate at 37°C overnight.

11. Select colonies and analyze by plasmid isolation, PCR, or sequencing. 

NEB® 5-alpha Competent E. coli (High Efficiency)

1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.

2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 minutes. Do not mix.

4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.

5. Place on ice for 5 minutes. Do not mix.

6. Pipette 950 µl of room temperature SOC into the mixture.

7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

8. Warm selection plates to 37°C.

9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

Mouse Genotyping

Mouse Genotyping protocol

Step 1: Gather Materials and Reagents

• Make sure you have all the necessary materials and reagents ready for the genotyping process. This includes the tissue samples (tail snips, ear punches, or other tissues), Extraction Reagent, Stabilization Buffer, PCR reagents, and agarose gel.

• AccuStart™ II Mouse Genotyping Kit (Cat. No. 95135-100; Size: 100 reactions; Quantabio)

Step 2: Prepare Tissue Samples

• Depending on the type of tissue samples you have (tail snips, ear punches, or other tissues), determine the appropriate volume of Extraction Reagent needed. Refer to the table provided earlier for guidance on sample sizes and Extraction Reagent volumes.

• Ensure that tissue samples are small and completely submerged in Extraction Reagent.

Sample Type	Sample Size	Extraction Reagent Volume	Comments
Tail Snips	2 mm	100 μL	Fresh or frozen tail snips can be used. Use proportionally more Extraction Reagent with tail snips larger than 2 mm. Samples will appear to remain intact and will not dissolve in Extraction Reagent after heating.
Ear Punches	2 mm	50 μL	Ensure that the ear punches are completely submerged in Extraction Reagent.
Other Tissues	5 mg	100 μL	Tissue samples should be small and completely submerged in Extraction Reagent.

Step 3: DNA Extraction

• Add the tissue samples to the appropriate volume of Extraction Reagent as calculated in Step 2.

• Heat the samples to 95°C for 30 minutes. Note that the tissue samples will not dissolve and will appear intact after incubation.

• Cool the samples to room temperature.

• Add an equal volume of Stabilization Buffer to the cooled samples. This serves as a safe stopping point.

• Extracts can be stored at 4°C for several weeks or at -20°C for several months.

Step 4: PCR Reaction Setup

• Use up to 2.5 μL of the DNA extract in a 25 μL PCR reaction. Depending on the sample size and extraction conditions, you may need to dilute the extract in water or TE buffer for optimal PCR results.

• Follow your standard PCR reaction setup protocols for amplifying the DNA regions of interest.

Step 5: Gel Electrophoresis

• Load 5 or 10 μL (or as appropriate) of the PCR products on an agarose gel.

• Perform gel electrophoresis to visualize and confirm the presence of the desired DNA fragments.

Additional Notes:

The protocol can be optimized for specific applications by adjusting incubation times or omitting the addition of Stabilization Buffer if needed.

It's recommended to use PCR tubes or multi-well plates and incubate in a thermal cycler with a heated lid to prevent sample condensation.

Complementary DNA Sequence Generator

Description: The "Complementary DNA Sequence Generator" is a web-based tool that allows users to generate the complementary DNA (cDNA) sequence of a given DNA sequence. It is a simple and user-friendly utility designed to assist in molecular biology and genetics research. Users can follow these steps to utilize the tool:

Usage Instructions:

1. Enter your DNA sequence in the input field above.
2. Click the "Generate" button to create the complementary sequence.
3. The complementary sequence will be displayed below.

Enter DNA Sequence:

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Complementary Sequence:

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