Help restriction analysis of plasmid - (Jun/10/2008 )
I am a novice in cloning reaction, and I am trying to clone gene of 1.1kb into pET-100/D-TOPO (invitrogen).
PCR was done by using AccuPrime Pfx super mix and primers designed and genomic DNA. After the clone reaction, I find several colonies on my plates (LB+Amp). I cultured the colonies from my plates overnight, and purified plasmid using purification kit.
To confirm the insert, restriction digestion was done by using Hind III. Hind III should cut both vector and insert once. So, I suppose to find one band of 5.6 kp and one band of 1.2 kp on the gel. But, I found one band of 5.6 kp and another band of 2.4 kp on my gel.
What could be wrong in my experiment? Thanks for any suggestion.
PCR was done by using AccuPrime Pfx super mix and primers designed and genomic DNA. After the clone reaction, I find several colonies on my plates (LB+Amp). I cultured the colonies from my plates overnight, and purified plasmid using purification kit.
To confirm the insert, restriction digestion was done by using Hind III. Hind III should cut both vector and insert once. So, I suppose to find one band of 5.6 kp and one band of 1.2 kp on the gel. But, I found one band of 5.6 kp and another band of 2.4 kp on my gel.
What could be wrong in my experiment? Thanks for any suggestion.
Sherry, cloning is a basic technique but without experience there are about 542 things that can go wrong.
Discuss your entire technique with a senior colleague, tell him/her every single step in detail,
show gel photographs,
controls,
check plasmid,
sequence,
MCS,
antibiotics,
check reagents names,
expiry dates,
temperatures of equipment,
and all the while, read more about cloning. Some here:
http://search.vadlo.com/b/q?sn=158621799&a...oning&rel=0
http://search.vadlo.com/b/q?sn=158621799&a...ning+&rel=2
That is the best way to go about cloning in the beginning.
In fact, it is good that you are facing troubles, you will be an expert in no time!
Hi
Have u check your insert for any internal restriction site for Hind III and as i dont have any information about your vector, insert and ligation condition, i have another doubt of self ligation of vector and insert (1vector both end ligation and forming circle this can give a band of 5.6 kb after restriction and 2 insert ligated to each other forming circle can give 2.4 (1.2+1.2) band on restriction.
Have u check your insert for any internal restriction site for Hind III and as i dont have any information about your vector, insert and ligation condition, i have another doubt of self ligation of vector and insert (1vector both end ligation and forming circle this can give a band of 5.6 kb after restriction and 2 insert ligated to each other forming circle can give 2.4 (1.2+1.2) band on restriction.
Yes, my insert has a restriction site for Hind III. I assume if the circle formed by two inserts can still be cut into 1.2kb, am I right?
Hi Sherry_Tong.
Would you mind to post up the gel photo?Also, the RE manufacturer, digestion condition is needed to troubleshoot it.
Personally, i think self-ligation happens at very low level that is hardly to be detected. You didn't add in ligase did you? 
Try to do double digestion with different RE.
But 2.4k bp is the multiple of 1.2k bp...Is there any relationship between this two sizes?Hmm...looking for answer from expert 
Would you mind to post up the gel photo?Also, the RE manufacturer, digestion condition is needed to troubleshoot it.
Personally, i think self-ligation happens at very low level that is hardly to be detected. You didn't add in ligase did you?
Try to do double digestion with different RE.
But 2.4k bp is the multiple of 1.2k bp...Is there any relationship between this two sizes?Hmm...looking for answer from expert
Hind III and Reaction buffer are from Invitrogen. I used 5 unit/10 ul for my restriction reaction for 1 hour at 37 oC. I did not add in ligase during restriction analysis and clone reaction.
Thanks for your reply..
Have u check your insert for any internal restriction site for Hind III and as i dont have any information about your vector, insert and ligation condition, i have another doubt of self ligation of vector and insert (1vector both end ligation and forming circle this can give a band of 5.6 kb after restriction and 2 insert ligated to each other forming circle can give 2.4 (1.2+1.2) band on restriction.
Yes, my insert has a restriction site for Hind III. I assume if the circle formed by two inserts can still be cut into 1.2kb, am I right?
can u give details about at what position vector and insert has site for Hind III.
Have u check your insert for any internal restriction site for Hind III and as i dont have any information about your vector, insert and ligation condition, i have another doubt of self ligation of vector and insert (1vector both end ligation and forming circle this can give a band of 5.6 kb after restriction and 2 insert ligated to each other forming circle can give 2.4 (1.2+1.2) band on restriction.
Yes, my insert has a restriction site for Hind III. I assume if the circle formed by two inserts can still be cut into 1.2kb, am I right?
can u give details about at what position vector and insert has site for Hind III.
Hind III cuts vector at 709, and cuts my insert at 189. I also tried another RE Acc I, which should also cut vector and my insert both once. I had the exactly same bands on my gel!!!
That was a good idea Sherry - to try another digest. Try another couple, but this time use a combination of restriction enzymes (in separate reactions) that will:
a. digest the insert and on both sides of the vector - this will provide you with more information about the insert.
b. only digest outside the insert - if you have a dimer you will see twice the size of the insert band you expect
Have u check your insert for any internal restriction site for Hind III and as i dont have any information about your vector, insert and ligation condition, i have another doubt of self ligation of vector and insert (1vector both end ligation and forming circle this can give a band of 5.6 kb after restriction and 2 insert ligated to each other forming circle can give 2.4 (1.2+1.2) band on restriction.
Yes, my insert has a restriction site for Hind III. I assume if the circle formed by two inserts can still be cut into 1.2kb, am I right?
can u give details about at what position vector and insert has site for Hind III.
Hind III cuts vector at 709, and cuts my insert at 189. I also tried another RE Acc I, which should also cut vector and my insert both once. I had the exactly same bands on my gel!!!
what is the total size of vector and insert ?