Digestion Ligation (DigLig) LI and LII
Introduction
Materials
- MQ
- 10X Tango Buffer
- ATP
- T4 DNA Ligase
- Enzymes
- BSA
- T4 Buffer (as an alternative to 10X Tango Buffer and ATP)
- Plasmids
- Backbone and insert
Procedure
How to Calclulate the Concentrations in the case of our MFalpha:
Go to this website and type in the DNA concentrations and bp lengths that you have for insert and vector, choose 2:1 ratio. So in this example we need around 13ng of insert.
Note: For LII assembly, You have to put in the bp length of the whole plasmid and not that of the insert it contains.
- The length of our Mfa is 317bp (including the amplification overhangs) and we choose (2:1) ratio, so you need 12.8ng of insert Mfa.
- Nanodrop your insert: we have 68.5ng/µL Mfa
Calculate how much of the stock DNA solution needs to be added. in the case of MFa: (12.8ng)/(68.5ng/µL)=0.19µL. This is too small a volume to pipette directly. If this happens, you need to dilute it (for example, 1.9µL of the insert in 8.1µL of MQ) This would yield a solution with MFa concentration of 12.8ng/µL so you could just add 1µL of this dilution to your reaction to add 12.8ng. Use the formula c1xV1=c2xV2 to dilute your inserts.
V1=amount of stock you add to dilution
C1= conc. of stock (NANODROPED)
V2= final volume= added stock plus MQ to dilute (10µL)
C2 = conc. of dilution (=required insert DNA mass)
V1 uL + ( 10µL-V1 uL) uLMQ= dilution (total volume of 10µL)
A A | B B | Example for MFa in p02 Example for MFa in p02 | |
1 1 | H20 | fill up to 15 ul | 10.5 μl |
2 2 | Vector (7.5 nM) | calculate as described above | dilute 1 ul in 3 ul MQ, then add 1 ul |
3 3 | Insert (15 nM) | calculate as described above | dilute 1.9 ul in 8.1 ul MQ, then add 1 ul |
4 4 | 10X Tango Buffer | 1 μl | 1 μl |
5 5 | T4 DNA Ligase (5 units/μl) | 0.25 μl | 0.25 μl |
6 6 | Enzyme (5 units/μl) | 0.25 μl | 0.25 μl |
7 7 | ATP (10 mM) | 1 μl | 1 μl |
A A | B B | |
1 1 | H20 | fill up to 15 ul |
2 2 | Vector (7.5 nM) p(10) for all! | calculate as described above |
3 3 | Insert A-B Promoter (15 nM) | calculate as described above |
4 4 | Insert B-C MFa or dummy BB6 for B+ (15 nM) | calculate as described above |
5 5 | Insert C-D Receptor (15 nM) | calculate as described above |
6 6 | Insert D-E Fluorescent protein(15 nM) 125.9 ng (2:1) | calculate as described above |
7 7 | Insert E-F Terminator (15 nM) | calculate as described above |
8 8 | Insert F-G BB9 dummy (15 nM) | calculate as described above |
9 9 | 10X Tango Buffer | 1 .5 μl |
10 10 | T4 DNA Ligase (5 units/μl) our tube has 400000 u per ml-> 400u/uL | 0.25uL |
11 11 | Enzyme (5 units/μl) | 0.25 μl dilution: (1uL EZ in 3uL MQ =to volume 4uL) -> then add 1uL |
12 12 | ATP (10 mM) | 1 μl |
- The program on the thermocycler is diglig long, which can also be used for short insert as well as for the LII constructs.
Digestion Ligation is a procedure in restriction cloning and it is the procedure with which we assemble the constructs in our iGEM project. ALWAYS: when we do DIGLIG, which needs 4h time then first check if one of the small blocks A or B is available! In this protocol, we use the insertion of MFa into the p02 Vector as an example