Agaroz Jel Elektroforezi

A-Agaroz Jel Hazırlanması

1. Gerekli miktarda agaroz tartılır, erlene koyulur.
2. Gerekli miktarda TBE eklenir.
3. Hafifçe çalkalanıp mikrodalga fırına koyulur.
4. Mikrodalga fırın örnek kaynamaya başlayana kadar yüksek ayarda çalıştırılır.
5. Aralıklarla erlen mikrodalgadan çıkarılıp çalkalanır.
6. Kaynama başladıktan sonra mikrodalganın ayarı düşürülür. Tamamen eriyene
kadar çalkalanarak ısıtılır.
7. EtBr eklenir ve çalkalayarak tüm çözeltiye dağılması sağlanır.
8. Trayin çatlamaması için çözelti muslukta biraz soğutulur.
9. Traye yavaşça dökülür ve polimerize olması için en az 15 dakika beklenir.
B- Örneklerin Jele Yüklenmesi
1. Jel tanka yerleştirilir.
2. Tank jelin üstünü kapatacak kadar TBE ile doldurulur.
3. Küçük bir parça parafilm alınır ve yüklenecek örnek sayısı kadar yükleme
tamponu (yaklaşık 1’er μl) üstüne koyulur.
4. 5 μl örnek yükleme tamponu ile karıştırılıp jele yüklenir.
5. Bir kuyucuğa da markır koyulur.
6. 80 V da 20 dakika yürütülür.
TBE Tamponu AppliChem TBE Buffer 10X A3945
Agaroz AppliChem Agarose Low EEO A2114
EtBr Sigma Ethidium Bromide Solution E1385
Bromfenol Mavisi Sigma Bromphenol Blue B0126
Tris Sigma Trizma base T6066
Gliserol Fluka Glycerol 49767 .................More Read....

Coomassie Blue Stain:  (for gels)
1) Combine 225 ml Methanol with 225 ml ddH2O.
2) Add 0.5 grams of Coomassie Blue.
3) Just before
use, add 50 ml acetic acid to 10% final concentration.
4) Stain for 2 hours.
Staining time can be shortened by microwaving; ~50 seconds at medium power.

5) After staining, pour off the stain into a bottle marked Used Stain.
Stain can be re-used once, but fresh 10% acetic acid must be
added).

Coomassie Blue Destain:  (for gels)

1) Combine 25 ml of Methanol with 437.5 ml ddH2O.
2) Just before use, add 37.5 ml acetic acid to 7.5% final
concentration.
3) Destain, plus sponges (pieces of foam packing) for a
couple of hours.  It may be necessary to use more than one change of destain.
Microwaving can be used in this stage also, but the details have not been
determined.

Coomassie Blue Stain:  (for membranes)

1) Combine 50 ml of Methanol with 50 ml of ddH2O.
2) Add 0.1 g of Coomassie Blue.
3) Stain by pouring a
sufficient amount to cover the membrane in a petri dish.
4) Wash the fluid
gentle back and forth over the membrane.  Allow it to be immersed for one to two
minutes.
5) Dispose of stain in a manner similar to that for the gel
stain.

Dialysis Tubing Prep
1) Cut tubing
to appropriate lengths and place them in a large beaker.
2) Add enough 5%
NaHCO₃ (add 50 grams per liter of ddH2O) to cover the tubing.
3) Bring to a rolling boil and allow
it to boil for 15 minutes.  The tubing will float so place a beaker of the next
smaller size on top of the tubing.
4) Rinse the tubing with an excess of
ddH2O.
5) Soak the tubing in 2 mM EDTA (add 10 ml 0.2 M
to 1 liter ddH2O) for one hour.
6) Rinse again with an
excess of ddH2O.
7) Store in 0.05% Azide (add 0.005 ml
per liter of ddH2O).

To Pour Stacking
Gel…
0.4mls  Acrylamide
0.4mls  10x
Stacking Buffer
40uls  10% SDS
3.12mls  ddH20
2uls  Temed
34uls  APS

How to Make 15%
Mini-gels
**Important** Due
to the fact that Acrylamide is being used, it is extremely important to wear
gloves throughout this procedure.
1) Measure out 5.152-ml ddH2O
into a sterile 15-ml-tube.
2) Add 4.58-ml Acrylamide.
3) Next add 1.1-ml
10x Seperating Buffer.
4) Then add 110-ml 10% SDS.
5) Continue by adding
56-ml APS.
6) Finally, add 5.6-ml Temed.(Note: Temed is the polymerizing
agent so the following steps must be done fairly quickly.)
7) Use a
disposable pipet, homogenize the solution being careful not to cause any
bubbles.
Then use the same pipet to fill the glass leaving about 1 inch
at the top.
9) Finally, add 0.5 inch of H2O-saturated
butanol on top of the gel. Note: Leave the unused portion of the solution in the
15-ml tube. Use this to determine if your gel is ready to use (when the tube is
polymerized, so is your gel).
.................More Read....