January 31, 2011

We come back from Christmas Holidays:

-We revise again all the reactive and we determine which one was missing
-We concentrate the DNA that we have obtained from the last experiment

DNA concentrations determined:

         6 Rinse= 40ug/ul

         5 (2)= 60mg/ul

December 22, 2010

We can't realize the End- repair protocol because we don't have the enough End- Repair reactive

December 21, 2010.

ELECTROELUTION CONCENTRATIONS:

5=165ng/ul

5Rinse=40ng/u

6=115ng/ul

6Rinse=125ng/ul

December 1, 2010.

ELECTROELUTION

-We had substitute the Agar ACE with GELase
-4˚C Overnight

DNA concentrations:

S1=45ng/ul

S2=145ng/ml

November 3, 2010.

Oct. 26, 2010. SIZING GEL Modifications: Ladders: 40kb and Lambda Hind III

5= Bottle 5

6= Bottle 6

 L1= Hind III

L2= 40kb

October 27, 2010.

SIZING GEL Modifications: Ladders: 40kb and Lambda Hind III

1= Bottle 1

2= Bottle 2

 L1= Hind III

L2= 40kb

3= Bottle 3

4= Bottle 4

September 29, 2010.

Chaotropic buffer substitution:

-Guanidine isothiocyanate (GITC) with Guanidine HCl

                G-HCl 5M in 20ml

October 4, 2010.

Bottles	Soil (g)	Z Buffer (ml)
1	15.028g	22.5ml
2	15.12g	22.5ml
3	15.13g	22.5ml
4	15.05g	22.5ml
5	15.07g	22.5ml
6	15.05g	22.5ml

**Freeze and Thaw**

October 9, 2010.

DNA Purification

September 27, 2010.

First of all we optimize the protocol based on the amount of sample and other materials that we had for work.

-We prepared stocks of:

CTAB  100ml and EDTA  500ml

-Computations for use 50ml bottles for the Freeze and Thaw:

	Original	Modified	Total (min)
bottle	250ml	50ml
Soil	50g	15g	90g
Z buffer	75ml	22.5ml	135ml
SDS	9ml	2.7ml	16.2ml
GITC	4.5ml	1.35ml	8.1

·         The intention is to extract DNA from soil using three 50ml bottles with 15g of soil in each one.

We will have:

6 bottles=90g of soil

September 28, 2010.

-We prepared stocks of:

NaCl (5M)

Sodium Phosphate solution

Tris HCl (1M)

Z Buffer

(100ml sterile Tris, 50ml sterile Sodium Phosphate Solution, 200ml sterile EDTA, 300ml sterile NaCl, 100ml CTAB 10%, 250ml Water dH₂O)

SDS 20 %

                We substitute the Lauryl with Sodium dodecyl sulfate