Solexa DNA Prep-2

skirlorangeΒιοτεχνολογία

1 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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1.

Extract Genomic DNA

2.

Shear Genomic DNA


1.

Place 1
-
3ug genomic DNA into a total volume of 200ul TE in a 1.5ml tube

2.

Sonicate using the following settings:



Duty Cycle:


80%



Output Control:

1.9



Sets of 20 pulses, a total of 7 rounds (place on ice to cool, then



centrifuge prior to next round)

3.

Concentrate DNA in Zymo Research DNA Concentrator kit. Elute in 6ul


water.


3.

End repair

(Epicentre End
-
IT Repair kit)


1.

Prepare the following reacti
on mix:




Eluted DNA





10
µL



H
2
O






24
µL



10X End
-
Repair Buffer



5
µL



dNTP mix






5
µL



ATP






5µL



End
-
Repair Enzyme Mix



1
µ
l






2.

Incubate the sample at
25°C
for
45 minutes.

3.

Purify the DNA with Zymo Research DNA Concentrator kit. Elute in 10ul
water.


4.

Addition of a single <A> base

(NEB)


1.

Prepare the following reaction mix:




Eluted DNA




10
µL



H
2
O





31
µL



Klenow buffer




5
µL



dATP(10mM)





1
µL



Klenow 3’ to 5’ exo
-

(5U/µL)


3
µL


2.

I
n
cubate the sample at 37°C in for 30min
.

3.

Purify the DNA with
Zymo Research DNA Concentrator kit
. Elute in 9
ul
water.






5.

Adaptor ligation (Epicentre Fast
-
Link DNA Ligation Kits)


1.

Prepare the following reaction mix:




Eluted DNA




9
µL



10X Fast
-
Link Ligation Buffer

1.5
µL



ATP (10mM)




1.5
µL



Adaptor oligo mix (4
5uM)


2
µL



Fast
-
Link
DNA
Ligase



1
µL


2.

Incubate at 25°C for 15 minutes.

3.

Purify the DNA with Zymo Research DNA Concentrator kit. Elute in 10ul
water.


6.

Gel purification of cDNA Templates (Zymo Research)


1.

Prepare a 1.5
% agarose gel in a final volum
e of
100mL 1x TBE

buffer.

2.

Load the 10µL
of sample into one well, and 3
-
5
µL of 100bp DNA ladder

(Fermentas
) into another well. (for handling mul
tiple samples, leave at
least 1 well

between samples to prevent cross contamination)

3.

Run gel at 120V for 60
-
90min til
l sufficient separation of the 100bp and
200bp bands of the DNA ladder.

4.

Cut a gel slice at 200 bp (+/
-

25bp) to 500 bp as showing in figure 1, and
purify the DNA with Zymo Research Zymoclean Gel DNA Recovery Kit.
Elute the DNA into
10
µL

of water.

5.

***Note:
Melt gel at room temperature.




Figure
1
: A 1.5% Agarose gel showing the region that should be cut to purify DNA fragments for
library preparation.



7.


PCR

Enrichment of Purified cDNA Templates


1.

Set up PCR master mix, make 10
% extra reagent for multiple samples
:




5


cloned
Phu
sion

Buffer

HF



10µL




PCR primer 1.1




1
µL



PCR primer 2.1




1
µL



10
mM dNTP mix




1
.5µL



Phusion polymerase

(NEB, #F
-
530)


1
µL



H2O






25.5
µL

2.

Add 10
µL

of purified

ligation mix

from section 2.8, step 4 to the PCR
tube.



3.

Run following PCR cycle:



98°C

1 min



98°C

10 sec




65
°C

30 sec


15




72°C

45

sec





72°C

5

min



4°C

hold

4.

Purify the DNA with
Zymo Research DNA Concentrator kit. Elute in 26ul
Qiagen EB Buffer.


9.

10nM
Dilution

1.

Dilute the sample to 10nM scale:

1)

To do this calculation, we need to know the size range of the DNA
fragments and take the average of that range. For example, a library
with an average size of 250bp.

2)

We need to multiply this number by 650. If the a
verage size range was
250, the result of this multiplication is 162,500=(150)(650).

3)

Next we need to divide the nanogram/microliter DNA concentration
(measured by the user) by the result of (2). For example, if a user
measures a concentration of 20 nanogram
s/microliter, and their
average size was 250, the calculation would be 20/162500 which is
0.00012308.

4)

To convert the result of (3) to the concentration in nanomolar, we need
to multiply by 1,000,000. In this example this gives us a concentration
of 123.076
923 nanomolar

5)

If we divide the result of (4) by a number to produce a value of 10,
then in this case if we divide by 12.2, the resulting value is 10.08 nM.

6)

Take the value you divided by in (4), which for this case is 12.2 and
multiple that by the total vol
ume of your sample. Lets say 21ul. Then
this gives you (12.2)(21)=256.2. You now need to dilute your sample
to a final volume of 256.2ul in Buffer EB including 0.1% Tween 20.

7)

For this example:



Buffer EB

234.94ul



Tween 20

0.2562ul



Sample

21ul



Total

Vol.

256.2ul