Does this look alright? - (Mar/24/2014 )

Vector

1) Miniprepp

2) Measure DNA-concentration

3) Electrophoresis to validate vector is intact & right size

4) Digest the 5' and 3' with 2 restriction-enzymes

5) Run a electrophoresis with just a small sample (3mikroliters)

6) If electrophoresis shows successful digestion, then the whole sample can be used for the ligation

 

 

Insert

1) Miniprepp

2) Measure DNA-concentration

3) PCR (two dilutions and one control)

4) Electrophoresis to validate PCR

5) PCR purification

6) Measure DNA-concentration (how much DNA is it expected to loose here normally?)

7) Digest the 5' and 3' with 2 restriction-enzymes

8) Purify once again

9) Measure DNA-concentration for the ligation-calculations

 

Ligation

If everything is OK, then ligate the vector with my insert

 

 

My questions

 

Question 1)

Should I purify the vector-sample at some point? Maybe after the digestion? Or is it not necessary?

 

Question 2)

At what step should I measure the DNA-concentration of the vector? After the digestion (step 4)?

I need this concentration for calculating how much vector to go with insert for the ligation.

 

Question 3)

Do you guys always calculate the exact volumes necessary of vector and insert for the ligation? Or do you just go with:

- 2 microliters vector

- 1 microliters insert

- 2 microliters buffer

- 1microliter T4

- H2O up to a total volume of 10microliters

Purify only when absolutely necessary. That means after PCR (before digestion) and after PCR (unless you can heat kill, strongly recommended!), and after vector digestion (unless you can heat kill, strongly recommended!).

I never calculate ligation volumes -- you can't pipet that well in any case.

I would run samples of cut and purified insert and vector on a gel to verify presence and quantity.

Why are you adding 2 ul of the T4 ligase buffer in a 10 ul ligation -- it is a 10x buffer!

Question 1)

So I should run a small sample (3mikroliters) of both the vector & insert on a gel to verify presence and quantity. If the gel shows 1 strong nice band of each one, then I am good to go. Right?

 

Question 2)

First time I mixed the following for the ligation:

- 1ul vector (45ng/ul)

- 0.76ul insert (17ng/ul)

- 8.24ul H2O of a dilution buffer that was 1x (the 5x stock had been diluted down to 1x and from this 1X I took 8.24ul)

____________

a total volume of 10ul solution

To this I then according to the protocol added 10ul Ligation buffer and 1ul ligase.

But then I realised that by diluting the 5X down to 1X and using this 1X in a total volume of 10ul, will dilute it EVEN MORE!!!!

 

So this time, no matter what the concentrations I get from the vector and insert, I am thinking of doing the following:

- 1ul vector

- 2ul insert

- 2ul dilution buffer that is 5x

- 5ul H2O

-----------------------

If I am right in my calculations, this would yield me a total volume of 10ul that is 1x in dilution buffer (I need 1X!!!!!). And then I continue with the protocol and add 10ul ligase buffer and 1ul ligase.

 

Is all of this correct procedure?

 

Thanks phage434!!!!

 

 

phage434 on Mon Mar 24 17:02:08 2014 said:

Purify only when absolutely necessary. That means after PCR (before digestion) and after PCR (unless you can heat kill, strongly recommended!), and after vector digestion (unless you can heat kill, strongly recommended!).

I never calculate ligation volumes -- you can't pipet that well in any case.

I would run samples of cut and purified insert and vector on a gel to verify presence and quantity.

Why are you adding 2 ul of the T4 ligase buffer in a 10 ul ligation -- it is a 10x buffer!

1) Yes.

2) Sounds ok