DNA Cloning Protocol

[moleculardude]

DNA Cloning Protocol

Need:

Vector DNA

Vector DNA can be:
Vector or Plasmid DNA (which needs to be

To clone you need at least several micrograms (3-5 or more) of your vector DNA.


Insert DNA

Insert DNA can be:
Oligonucleotide Insert (oligos with restriction cut half-sites annealed and phosphorylated with PNK)
Restriction Enzyme Generated Insert
PCR Insert (PCR with restriction enzyme cut sites (the whole site is needed!) in the 5’ end of the primer)

If the Insert you want to clone is less than 60 bp, you can order oligonucleotides and anneal them together.

If your insert DNA size is small (less than 1000 bp), you do not theoretically need as much DNA as your vector. If your insert DNA size is tiny (10-200 bp), you need very little of this DNA theoretically.

In general you should have at least 1 microgram of starting insert DNA.

Preparing Vector DNA for Cloning : Mini-prep, Midi-prep, Maxi-prep

We found that the easiest way to prepare DNA is using a mini-prep kit (Qiagen). We use several columns for the same vector DNA construct.

For each column we add about 4 mL of LB grown bacteria (we add 2 mL of LB bacteria broth to a 2 mL eppendorf tube and spin twice = 2 X 2 mL = 4 mL)

Tip: Always grow about 5 mL of LB in 15 mL Falcon Tubes, keep the 1 mL as a glycerol bacteria stock frozen in the – 80 C freezer.

How to make a Glycerol Bacterial Stock for the – 80 C Freezer which will keep for Years (you will never have to streak plates or make competent cells again! – saves you time)

Simply add 50% glycerol to your bacterial growth LB. This does not have to be exact ie if you are left with about 1 mL of bacterial LB add about 500 uL of 100% glycerol and freeze in the – 80 C.

After eluting each column with about 50 uL of EB (elution buffer, Qiagen, Tris buffer – does NOT contain EDTA), we pool the eluates such that we have around 200 uL to 400 uL (or 4 – 8 columns per construct or vector). This is gives enough starting vector DNA for cloning.

Note: we found that the EDTA in TE inhibits subsequent restriction digestion of the eluted DNA from mini-prep and other plasmid purification kits.


Preparing Insert DNA for Cloning

Insert DNA must be


Restriction Enzyme Digestion and Cutting Your Plasmid Vector for DNA Cloning

Plasmid vector is usually cut.



Ligation of Insert and Vector

To Ligate Insert to Vector, use 200 ng of Vector as a guide (people use as little as 50 ng – N.E.B manual).



Insert ng = Vector ng
Insert size Vector size


Therefore to ligate a 500 bp insert into a 5000 bp vector you would need

Insert ng = 200 ng X 500
5000

Insert ng = 20 ng of insert needed for a 1 : 1 ligation

For a insert 3 : 1 vector ligation you would need: 60 ng



Ligation

X uL DNA vector 200 ng
Y uL Insert (calculated ng)
2 uL 10 X Buffer
1 uL T4 DNA Ligase NEB
to 20 uL H20
--------
20 uL Total

Take 5 uL of Ligation and add to 100 uL of DH 5 alpha cells Invitrogen
(Subcloning efficiency ok, One Shot better, Max Efficiency best – for hard ligations but expensive).

Heat Shock
Add 250 uL of SOC to competent Cells


Plate ENTIRE amount on plate.

Grow overnite at 37 C

[lei06]

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[rida_e_noor]

thanx dear..
keep sharing ...