Purification and storage of T-vector. 1) Purify the T-tailed DNA solution either using column or bu Phenol/Chloroform/Ethanol precipitation. 2) In a 1.5 mL ...
Incubate 2-3 hours at room temperature or overnight at 16-18°C. Experiments have been done showing that after 2 hours 95% of the product obtain after overnight ligation was obtained.
Molecular ratio calculation:
minsert =
size insert x m vector size vector
The amount of insert to add is a multiple of this ratio.
Molecular cloning 4
Molecular cloning
Ligation of double-stranded oligonucleotides
1) In a 1.5 mL Eppendorf tube, mix: - 2 µL of each oligonucleotide (100 µM) - 2 µL of 10X denaturation Buffer - 14 µL ddH20 2) Seal the tube with parafilm and place it in a Becher full of tap water 3) Heat the water until it starts to boil 4) Remove it from the microwave and keep the tube immerged. 5) Let cool at room temperature 6) For ligation mix: -
1 µL of 10X buffer
-
2 µL of 5X PEG8000 solution
-
10-15 ng of vector
-
1 µL of ligase
-
H2O up to 10 µL
7) Incubate at room temperature for 2 hours 8) Transform 2 µL of ligation
Denaturation Buffer (10X) 500 mM Tris-HCl pH = 7.4 100 mM MgCl2
Molecular cloning 5
Molecular cloning
Universal T/A cloning T-vector preparation
Purification of blunt-ended plasmid and addition of T-overhang 1) 1- In a 1.5 mL microcentrifuge tube, digest 10µg of plasmid with either - a blunt-end restriction enzyme (e.g. EcoRV) [Preffered] or - a 5’ overhang-end restriction enzyme filled-in with Klenow polymerase Mix by gentle vortex and centrifuge briefly. 2) Incubate at 37°C for 1-2 hours. 3) Run 1-2 µL on gel to ensure complete digestion. 4) Add 2 µL of 0.5 M EDTA to the tube at the end of digestion. 5) Purify the vector using on Qiagen column (e.g. Quiaquick PCR Purification Kit) 6) In a 1.5 mL microcentrifuge tube, add: - 46 µL of blunt-end digested plasmid - 15 µL of 5x TdT Buffer - 7.5 µL of CoCl2 - 1.5 µL of 1 mM ddTTP - 5 µL of Terminal Transferase (25 U/µL) 7) Mix gently and centrifuge briefly and incubate 1.5 hours at 37°C. The use of ddTTP prevents extension of a T-tail at the end of the DNA molecule. This could not be achieved with dTTP.
Purification and storage of T-vector 1) Purify the T-tailed DNA solution either using column or bu Phenol/Chloroform/Ethanol precipitation. 2) In a 1.5 mL microcentrifuge tube, add: - 44µL of purified plasmid as above - 5 µL of 10X ligase buffer - 3 µL of T4 DNA Ligase (1 U weiss/µL) Mix well and centrifuge briefly. Incubate overnight at 16°C. This step is not absolutely required as T/A cloning will be efficient at 70% without any further purification. If the vector used does not allow blue/white screening after cloning, ligation/gel purification increases T/A cloning efficiency to 90%.
Molecular cloning 6
Molecular cloning 3) Run a 1% agarose gel in TAE at 5V.cm-1 4) Excise the band with a scalpel blade and gel purify it (QuiaQuick Gel Exctraction Kit) The vector molecules on which a single T or no T was added will either form concatemers or self-ligate. Their size will be different from that of linear T-vector that has a T at both extremities. 5) Quantify purify T-vector and aliquot it at 25-30 ng/µL. Usually, ligation are done with 50/60 ng of vector.
Jan 1, 1995 - max. 10 mA. D.C. source or sink current into any output. ± IO max. 25 mA. D.C. current into the supply terminals. ± I max. 100 mA. PARAMETER.
Jan 1, 1995 - D.C. current into any input. ± II max. 10 mA. D.C. source or sink current into any output. ± IO max. 25 mA. D.C. current into the supply terminals.
Feb 5, 2010 - (Remark: for d = 1 this is easy; for d > 1 this exercise is a bit technical in the .... For example, (5.3.5) has mp = 1, (5.3.6) has mp = 2. Practical.
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