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Team:Heidelberg/AwesomeSheet/Enzymes
From 2014.igem.org
Lab book
– Enzymes
| A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | ID | Name | Concentration | Buffer | Inkubation | Inaktivation | max DNA conc. | Comment | BSA | ATP | Unit Defnition | |||||||||||||
2 | correct use | X | Name | X | T | t | T | t | ||||||||||||||||
3 | c/(u/µL) | /((U/uL)/(ng/uL)) | °C | min | °C | min | c/(ng/ul) | 1.1 | 2.2 | 3.1 | CutSmart | T7 Lig Buffer | ||||||||||||
4 | E | EcoRI-HF | 20 | 0.04 | 37 | 15 | 65 | 20 | 25 | 0 | 0% | 100% | 0% | 100% | One unit is defined as the amount of enzyme required to digest 1 μg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. | |||||||||
5 | X | XbaI | 20 | 0.04 | 37 | 15 | 65 | 20 | 25 | 0 | 0% | 100% | 75% | 100% | One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-/HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl. | |||||||||
6 | S | SpeI-HF | 10 | 0.02 | 37 | 15 | 65 | 20 | 25 | nur 5 x overdigestion | 0 | 75% | 100% | 25% | 100% | One unit is defined as the amount of enzyme required to digest 1 µg of Adenovirus-2 DNA in 1 hour at 37°C in a total reaction volume of 50 µl. | ||||||||
7 | P | PstI-HF | 20 | 0.04 | 37 | 15 | 65 | 20 | 25 | 0 | 10% | 75% | 50% | 100% | One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-/HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl. | |||||||||
8 | T4L | T4 DNA Ligase | 400 | 2 | 10xT4LBuffer | 10 | 16 | 10 | 65 | 10 | 10 | 1 | One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer. | The unit definition uses 0.12 μM (300 μg/ml) lambda HindIII fragments. The high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Vector:Insert molar ratios between 1:1 and 1:10 are recommended (1:3 is typical). If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. | ||||||||||
9 | T4LHC | T4 DNA Ligase h.c. | 2000 | 2 | 16 | 10 | 65 | 10 | 10 | 1 | One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer. | 300 | ||||||||||||
10 | T7L | T7 DNA Ligase | 3000 | 15 | 2xT7LBuffer | 2 | 25 | 30 | do | not | 10 | needs purification before electroporation | 1 | 1 | One unit is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 25°C in 1X T7 DNA Ligase Reaction Buffer. | 5 | ||||||||
11 | T7Lv | T7 DNA Ligase | 400 | 15 | 25 | 30 | do | not | 10 | needs purification before electroporation | 1 | 1 | One unit is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 25°C in 1X T7 DNA Ligase Reaction Buffer. | |||||||||||
12 | Flex MM | Phusion Flex Mastermix | 2 | no | no | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluBIe material in 30 minutes at 74°C | ||||||||||||||||||
13 | OneTaq | OneTaq Master Mix | 2 | never | ever | |||||||||||||||||||
14 | Flash MM | Phusion Flash Master Mix | 2 | |||||||||||||||||||||
15 | K | KpnI-HF | 20 | 0.04 | 37 | 15 | not in your | wildest dreams | 25 | 100% | 25% | 5% | 100% | |||||||||||
16 | BsaI | BsaI | 10 | 0.04 | 37 | 60 | 65 | 20.00 | 1 | 75% | 75% | 100% | 100% | |||||||||||
17 | BsmBI | BsmBI-HF | 10 | 0.04 | 55 | 15 | 80 | 20 | 25 | 0 | 10% | 50% | 100% | 25% | One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 55°C in a total reaction volume of 50 µl. | |||||||||
18 | BglII | BglII | 10 | 0.04 | 37 | no | pe | 10% | 10% | 100% | 10% | |||||||||||||
19 | XmaI | XmaI | 10 | 0.04 | 37 | 15 | 65 | 20 | 25% | 50% | 10% | 100% | One unit is defined as the amount of enzyme required to digest 1 µg adenovirus-2 in 1 hour at 37°C in a total reaction volume of 50 µl. | |||||||||||
20 | BsaI-HF | BsaI-HF | 20 | 0.04 | 37 | 15 | 65 | 20 | 1 | 50% | 100% | 25% | 100% | |||||||||||
