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From 2010.igem.org

CHECK-LIST PROCEDURE
Add;
- uL nuclease free water
- uL rapid DNA ligation buffer
- uL T4 DNA ligase
- uL from 25uL insert1(downstream) elute (elution must be done with nuclease free water)
- uL from backbone
- uL from 25uL (upstream) elute (elution must be done with nuclease free water) to end up in 15 uL T4 DNA ligase solution
- Keep the T4 DNA ligase solution in room temperature for 5 min.

NOTES
- After gel extraction, treatment with nuclease free water is needed.
- Total mixture shouldn’t exceed 15uL.
- Ligation ratios are calculated by comparing the molar ratios. Molar ratios of insert to vector is calculated by applying vector as reference.
Example: Vector ~ 2000 bp - Molar ratio is given as 1x

        
Insert 1 ~ 500 bp - Molar ratio is 4x compared to vector
        
Insert 2 ~ 100 bp - Molar ratio is 20x compared to vector

- At ligation, all inserts are put in 1x concentration
- Before ligation, nanodrop results are very significant. By these results, we can estimate the insert amount after gel extraction.
Example: Vector ~ 2000 bp at 2400 bp

        
Insert 1 ~ 600 bp at 2500 bp
        
Insert 2 ~ 100 bp at 2400 bp
  
If we start ligation with 5 ug each, after gel extraction, we will have appr. 4.0 ug Vector, 1,2 ug insert 1 and 200 ng insert 2.

- While estimating recovery from gel, always take account 10% loss of sample.
- With increasing restriction digestion volume, add 0.5 uL FD enzyme for 30-40 uL increasing of total volume. But add 1 uL enzyme for 1 ug plasmid DNA until total volume reaches to 50 uL.