Weird migration on gel - (Jan/08/2009 )
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
-jiajia1987-
QUOTE (jiajia1987 @ Jan 8 2009, 02:29 PM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have you done a PCR on your colonies to see if you have the correct size ?
Try to cut the plasmid only with one enzyme at the time and see what you get
-Jipes-
QUOTE (jiajia1987 @ Jan 8 2009, 05:29 AM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have a real good look at the DNA ladder designation for band weight.
send us gel pic to confirm.
thanks.
-Hanming86-
QUOTE (Jipes @ Jan 8 2009, 10:08 PM)
QUOTE (jiajia1987 @ Jan 8 2009, 02:29 PM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have you done a PCR on your colonies to see if you have the correct size ?
Try to cut the plasmid only with one enzyme at the time and see what you get
I did a miniprep for the ligated product colonies, and i sent it for sequencing. The first time I did it, there were no inserts at all.
I repeated the ligation today and I will be doing a sequencing reaction tomorrow, hopefully, I will be able to get some inserts.
My supervisor told us that one way to check if there is an insert in your ligation product would be to do a PCR. But I have not done that, because after all, I will be sending the minipreps of the ligated products for sequencing.
I have tried cutting the plasmid only with one enzyme, eg, Nde1, then do a PCR cleanup, after which I did a BamH1 digestion before I ran it on a gel. The results ended the same, everything were 3kb more than what it was supposed to be.
-jiajia1987-
QUOTE (Hanming86 @ Jan 8 2009, 10:38 PM)
QUOTE (jiajia1987 @ Jan 8 2009, 05:29 AM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have a real good look at the DNA ladder designation for band weight.
send us gel pic to confirm.
thanks.
I had a look at the DNA ladder designation for band weight, nothing is wrong. Everything is really 3kb heavier than they are supposed to be.
I will scan the gel results sometime tomorrow and send you for a look. How do I go about sending it to you? I am new here and I am not too sure of the functions.
-jiajia1987-
QUOTE (jiajia1987 @ Jan 8 2009, 07:04 AM)
QUOTE (Hanming86 @ Jan 8 2009, 10:38 PM)
QUOTE (jiajia1987 @ Jan 8 2009, 05:29 AM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have a real good look at the DNA ladder designation for band weight.
send us gel pic to confirm.
thanks.
I had a look at the DNA ladder designation for band weight, nothing is wrong. Everything is really 3kb heavier than they are supposed to be.
I will scan the gel results sometime tomorrow and send you for a look. How do I go about sending it to you? I am new here and I am not too sure of the functions.
Theres's an Attachment function. I can see it while writing this reply post. Just right underneath this reply box.
-Hanming86-
QUOTE (Hanming86 @ Jan 9 2009, 08:17 AM)
QUOTE (jiajia1987 @ Jan 8 2009, 07:04 AM)
QUOTE (Hanming86 @ Jan 8 2009, 10:38 PM)
QUOTE (jiajia1987 @ Jan 8 2009, 05:29 AM)
Hello all,
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
I started on my FYP recently and have encountered a problem for my gel electrophoresis.
My PCR product is supposed to be 1.5kb, but it shows up around 3kb more on the gel, which is 4.5kb. The funny thing is, when I do my plasmid digestion (Pet22b, which is around 5.5kb), I see the band at around 8Kb. After enzymatic digestion of the plasmid, the size of the plasmid will still be around 5Kb. I digested using Nde1 and BamH1.
The addition of 3kb is puzzling to me. I don't know why this keeps happening. I have done repeated plasmid digestion and used different ways of digeston, and the products always appear to be 3kb heavier. This also happens even when I repeat my PCR of my template and run a gel.
I ran the gels at 100V for 25mins using 1% agarose. The DNA ladder is 1kb plus DNA ladder.
I hope someone can provide me with some ideas. Thanks in advance!
Have a real good look at the DNA ladder designation for band weight.
send us gel pic to confirm.
thanks.
I had a look at the DNA ladder designation for band weight, nothing is wrong. Everything is really 3kb heavier than they are supposed to be.
I will scan the gel results sometime tomorrow and send you for a look. How do I go about sending it to you? I am new here and I am not too sure of the functions.
Theres's an Attachment function. I can see it while writing this reply post. Just right underneath this reply box.
Okay great. I saw it. Will scan and post tonight. Currently in the lab still. Thank you.
-jiajia1987-