Gibson Calculations Page

For Gibson Master Mix, use 3.75uL in a 5uL total volume (or 7.5uL in a 10uL total volume).

Calculating how much DNA to add to your Gibson Reaction:

NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0
pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases.

Use 75ng of Vector (50-100 ng is optimal)

Based on your DNA
concentration, figure out how many pmols/uL of Vector DNA you have (use [http://www.promega.com/a/apps/biomath/ promega
pmol calculator]).

Add 2-3x the number of
pmols for each of your fragments.

concentration: 100ng/uL (or .1ug/uL)

Using Promega:
0.025pmol/uL of DNA

Reaction:

Vector: 75ng x
(1uL/100ng) = 0.75uL

0.75uL x (0.025pmol/uL)
= 0.01875pmol of Vector

0.01875pmol of vector x
3 = 0.05625pmol of each fragment

Let’s say each fragment
is 500bp and 100ng/uL:

Using Promega: 0.3pmol/uL of DNA

0.05625pmol x
(1uL/0.3pmol) = 0.1875uL of DNA

We can’t accurately
pipette that small a volume, so dilute the DNA so that you are pipetting ~0.5uL
of each fragment, but still adding 0.05625pmol of DNA.

Here is our reaction:

Vector: 0.75uL
(.01875pmol)

Frag A: 0.5uL
(.05625pmol, which is 3xVector) after being diluted

Frag B: 0.5uL
(.05625pmol) after being diluted

**Now check your totals:**

Total number of pmol for
entire reactions should be between 0.02-0.5pmols: our total is 0.13pmol--PERFECT!

Total volume should be
2.5uL or under for a 10uL reaction and 1.25uL or under for a 5uL reaction. This
volume is 1.75uL, so we will opt for the 10uL reaction:

7.5uL of Gibson
Mastermix

0.75uL of Vector

0.5uL Frag A

0.5uL Frag B

Water up to 10uL, so
0.75uL of water.

Another Example:

25 February 2016 - LMF Gibson reaction, pET28a-StrepII-sfGFP-DmOptEnscAAs20-FLAG

SITUATION: One vector,
6151 bp, and one insert, 585 bp.

Vector starting
concentration: 105.5 ng/uL.

WANT: 75 ng of vector in
reaction.

***Vector***
calculations - [] in ng/uL to [] in pmol

1. 75 ng x (1

 uL/105 ng) = approx. 0.72 uL of
 vector - **add this to Gibson rxn.**

**How many pmol of vector**
does that mean we have?

2) Use pmol calculator
online: Length of vector = 6151 bp, concentration of vector = 0.105 ng/uL.

SO, **picomolarity of**
vector = 0.026 picomoles per microliter.

**3) We will add to rxn**: 0.72 uL of vector x 0.026 pmol/uL = **0.0187 pmoles of vector**

***Vector***
calculations - calculate from pmolarity of vector to uL of insert needed

Picomoles of insert
should be 2-3x picomoles of vector - we’ll go for 3x as much here, since we
have only 1 insert.

1. 0.0187

 pmoles vector x 3 = **0.056 pmoles of**
 insert needed

*(\*\*The insert was*
ordered directly. Need to calculate resuspension of geneblock. We received 500
ng of gene insert. If resuspend our insert in 10 uL, we will get a 50 ng/uL stock).

2) Use pmol calculator
online: Length of insert = 585 bp, concentration of insert = 0.05 ug/uL.

SO, **picomolarity of**
insert = 0.13 picomoles per microliter (pmoles/uL)

**3) We will add to rxn:** 0.056 pmoles insert x (0.13 pmoles/uL) = 0.43 uL.

***Create***
table to check volumes, picomolarity:

Volume    Picomoles

Vector
    0.72 uL        0.0187 pmoles

Insert
    0.43 uL        0.056 pmoles

**TOTAL:**
1.15 uL    0.0747 pmoles

**CHECK:**  Our
Gibson Master Mix is 1.3 x. We use 3.75 uL in a 5 uL reaction and 7.5 uL
in a 10 uL reaction. Thus, total volume of DNA added CANNOT exceed 1.25 uL for
a 5 uL reaction - we have 1.15 here, so this is a GO. Also, total picomoles of
DNA added should be between 0.02 and 0.5 picomoles - we are 0.0747 pmoles here,
so we are a GO.

Let’s change the volumes
slightly to get the exact 1.25 uL of DNA added. (Also, it is very hard to pipet
less than 0.5 uL, so this will be better for pipetting purposes too).

Volume    Picomoles

Vector
    0.75 uL        0.0187 pmoles

Insert
    0.50 uL        0.056 pmoles

**TOTAL:**
1.25 uL    0.0747 pmoles

**Check the math one more**
time and we are good to go!