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Team:METU Turkey/Results Discussion/LIGATION
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Cerenseref (Talk | contribs) (New page: <br>CHECK-LIST PROCEDURE <br>Add; <br>- uL nuclease free water <br>- uL rapid DNA ligation buffer <br>- uL T4 DNA ligase <br>- uL from 25uL insert1(downstream) elute (elution must be d...) |
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| - | <br>CHECK-LIST PROCEDURE | + | <br><h3>CHECK-LIST PROCEDURE</h3> |
<br>Add; | <br>Add; | ||
<br>- uL nuclease free water | <br>- uL nuclease free water | ||
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| - | <br>NOTES | + | <br><h3>NOTES</h3> |
<br>- After gel extraction, treatment with nuclease free water is needed. | <br>- After gel extraction, treatment with nuclease free water is needed. | ||
<br>- Total mixture shouldn’t exceed 15uL. | <br>- Total mixture shouldn’t exceed 15uL. | ||
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<br>Insert 1 ~ 600 bp at 2500 bp | <br>Insert 1 ~ 600 bp at 2500 bp | ||
<br>Insert 2 ~ 100 bp at 2400 bp | <br>Insert 2 ~ 100 bp at 2400 bp | ||
| - | <br>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. | + | <br>If we start ligation with 5 ug each, after gel extraction, we will have appr. 4.0 ug Vector, 1,2 ug insert 1 <br>and 200 ng insert 2. |
<br>- While estimating recovery from gel, always take account 10% loss of sample. | <br>- While estimating recovery from gel, always take account 10% loss of sample. | ||
<br>- 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. | <br>- 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. | ||
Latest revision as of 03:38, 28 October 2010
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.
