Southern Blot Questions - Optimal Ladder and Other Conditions (May/24/2010 )
Hello,
I have two questions about running southern blots.
1. I run my 0.8% gel (18 cm length, 350 ml total volume) at 20V overnight. I load 7.5 ul of 1 Kb DNA ladder from Invitrogen (cat. number 15615-016) and use 2.5 ul of 5X Loading Dye. However, I always get a smear with my ladder. Are there any recommendations in how to improve this?
2. After the gel-run, I do a 10 minute wash in 0.25M HCl, then two 3-5 min. washes in dH20, and then 30 min in 0.4M NaOH + 1mM EDTA. Are these conditions appropriate for optimal transfer? I do not know the exact rationale behind the HCl wash for 10 minutes or why the concentration of NaOH is 0.4M for denaturation - any help is appreciated.
3. What does 2X SSC buffer do to the blot?
Thanks!!
hotstuffdb22 on May 24 2010, 01:39 PM said:
I have two questions about running southern blots.
1. I run my 0.8% gel (18 cm length, 350 ml total volume) at 20V overnight. I load 7.5 ul of 1 Kb DNA ladder from Invitrogen (cat. number 15615-016) and use 2.5 ul of 5X Loading Dye. However, I always get a smear with my ladder. Are there any recommendations in how to improve this?
2. After the gel-run, I do a 10 minute wash in 0.25M HCl, then two 3-5 min. washes in dH20, and then 30 min in 0.4M NaOH + 1mM EDTA. Are these conditions appropriate for optimal transfer? I do not know the exact rationale behind the HCl wash for 10 minutes or why the concentration of NaOH is 0.4M for denaturation - any help is appreciated.
3. What does 2X SSC buffer do to the blot?
Thanks!!
Hi,
I run a similar sized gel, but only 250ml volume, and run at 200V for about 3hrs - until the dye band is approx 2cm from the bottom. I don't have any problems in the ladder amearing.
Do you run the gel in 1xTAE? this is the preferred buffer.
The HCL wash causes the DNA to fragment and allow better transfer.
The recipe for denaturation buffer I use is: 0.5M NaOH & 1.5M NaCl. Which I believe is fairly standard.
Followed by a neutralisation step; buffer recipe is 0.5M TRis pH to 7.5, 3M NaCl.
Hope this helps
Hello - Thanks for the response.
I run in 1x TBE, which I don't think is a huge difference.
So how long do you incubate in each buffer before transfer?
Also, you do the HCl, then the denaturation buffer, followed by the neutralization step. Do you use the neutralization buffer as the overnight transfer buffer?
wbone001 on May 24 2010, 10:03 AM said:
hotstuffdb22 on May 24 2010, 01:39 PM said:
I have two questions about running southern blots.
1. I run my 0.8% gel (18 cm length, 350 ml total volume) at 20V overnight. I load 7.5 ul of 1 Kb DNA ladder from Invitrogen (cat. number 15615-016) and use 2.5 ul of 5X Loading Dye. However, I always get a smear with my ladder. Are there any recommendations in how to improve this?
2. After the gel-run, I do a 10 minute wash in 0.25M HCl, then two 3-5 min. washes in dH20, and then 30 min in 0.4M NaOH + 1mM EDTA. Are these conditions appropriate for optimal transfer? I do not know the exact rationale behind the HCl wash for 10 minutes or why the concentration of NaOH is 0.4M for denaturation - any help is appreciated.
3. What does 2X SSC buffer do to the blot?
Thanks!!
Hi,
I run a similar sized gel, but only 250ml volume, and run at 200V for about 3hrs - until the dye band is approx 2cm from the bottom. I don't have any problems in the ladder amearing.
Do you run the gel in 1xTAE? this is the preferred buffer.
The HCL wash causes the DNA to fragment and allow better transfer.
The recipe for denaturation buffer I use is: 0.5M NaOH & 1.5M NaCl. Which I believe is fairly standard.
Followed by a neutralisation step; buffer recipe is 0.5M TRis pH to 7.5, 3M NaCl.
Hope this helps
Hi
The buffer may make a difference. I always understood that TAE was better at resolving larger fragments. My Southerns tend to be on genomic DNA and so have always used 1xTAE. May be worth a try.
I soak the gel in 0.25M Hcl for ~ 10 mins and then wash in dH2O for 2-3 mins. I usually wait for the blue dye front to turn yellow (This step is not always neccessary it eases transfer of large DNA fragments, > 15kb)
Denature for 2x 15mins, wash in dH2O for 5 mins
Neutralise for 2x 15mins, wash in dH2O for 5mins
all of the above is with gentle agitation.
I use 20x SSC in my overnight transfer - which is fairly standard - briefly soaking the membrane in 2xSSC
The SSC provides the high salt content that is needed to transfer the DNA.
Good luck!
W
hotstuffdb22 on May 24 2010, 09:10 PM said:
I run in 1x TBE, which I don't think is a huge difference.
So how long do you incubate in each buffer before transfer?
Also, you do the HCl, then the denaturation buffer, followed by the neutralization step. Do you use the neutralization buffer as the overnight transfer buffer?
wbone001 on May 24 2010, 10:03 AM said:
hotstuffdb22 on May 24 2010, 01:39 PM said:
I have two questions about running southern blots.
1. I run my 0.8% gel (18 cm length, 350 ml total volume) at 20V overnight. I load 7.5 ul of 1 Kb DNA ladder from Invitrogen (cat. number 15615-016) and use 2.5 ul of 5X Loading Dye. However, I always get a smear with my ladder. Are there any recommendations in how to improve this?
2. After the gel-run, I do a 10 minute wash in 0.25M HCl, then two 3-5 min. washes in dH20, and then 30 min in 0.4M NaOH + 1mM EDTA. Are these conditions appropriate for optimal transfer? I do not know the exact rationale behind the HCl wash for 10 minutes or why the concentration of NaOH is 0.4M for denaturation - any help is appreciated.
3. What does 2X SSC buffer do to the blot?
Thanks!!
Hi,
I run a similar sized gel, but only 250ml volume, and run at 200V for about 3hrs - until the dye band is approx 2cm from the bottom. I don't have any problems in the ladder amearing.
Do you run the gel in 1xTAE? this is the preferred buffer.
The HCL wash causes the DNA to fragment and allow better transfer.
The recipe for denaturation buffer I use is: 0.5M NaOH & 1.5M NaCl. Which I believe is fairly standard.
Followed by a neutralisation step; buffer recipe is 0.5M TRis pH to 7.5, 3M NaCl.
Hope this helps