Why is the fragment of insert shorter than I want? - (Dec/11/2006 )
Dear everybody!
I have tried to clone human insulin receptor gene for about 2 months. Now, I face a quesiton I can not realize. Is there anyone can help me?
First, I used nest PCR to amplify the insulin receptor gene from human cDNA library. The agatrose gel electrophoresis revealed probably correct PCR products I wanted. It was about 4kb in length. In addition to the product I wanted, in agatrose gel electrophoresis , there were several bands with different molecular weight. Hence, I cut the band with molecylar weight about 4kb and perform gel extraction. Then, I performed restriction enzyme digestion of the vector and PCR products. Thereafter, I performed ligation and used colony PCR to screen the positive colony. I could find few "positive colonies". Then, I done liquid culture and minipreparation. I cut the collected plasmid to check if it was a real positive colony. It was very strainge that I always found the band with size compatible with the vector but the another band was always shorter than 4kb. I even tried to do sequencing and found it was fragement of insulin receptor gene. What's the reason and how to resolve it?
I just made this very post in another thread a minute ago
I think that's the most likely explanation. You may also have a splice variant of your gene, although you probably should have seen that in the gel after you isolated your gene.
additional suggestion
is the fragment way shorter then 4kb is just a little shorter then 4kb (and thus could have been mistaken as 4kb during the initial gel extraction of the insert band)
Are all the clones shorten by the same amount. Could there be an unexpected RE site in the 4kb sequence. Some sequence polymorphism.
May be
1) your PCR product has an internal site for the Restriction Enzyme that you are using. If this site is very close to the end then you will not make out the difference on an agarose gel.
2) if there has been a mistake in your cutting the gel and you also purified some shorter PCR fragments, it is possible that you picked a colony arising out of the shorter insert ligating with your vector. this would be very rare as your insert will be dominated by the main band you cut out, but this is still a possibility.
I have tried to clone human insulin receptor gene for about 2 months. Now, I face a quesiton I can not realize. Is there anyone can help me?
First, I used nest PCR to amplify the insulin receptor gene from human cDNA library. The agatrose gel electrophoresis revealed probably correct PCR products I wanted. It was about 4kb in length. In addition to the product I wanted, in agatrose gel electrophoresis , there were several bands with different molecular weight. Hence, I cut the band with molecylar weight about 4kb and perform gel extraction. Then, I performed restriction enzyme digestion of the vector and PCR products. Thereafter, I performed ligation and used colony PCR to screen the positive colony. I could find few "positive colonies". Then, I done liquid culture and minipreparation. I cut the collected plasmid to check if it was a real positive colony. It was very strainge that I always found the band with size compatible with the vector but the another band was always shorter than 4kb. I even tried to do sequencing and found it was fragement of insulin receptor gene. What's the reason and how to resolve it?
Dear everybody!
I want to share my experiene with everyone. I think my competent cells might attack my insert. Actually, I ever screened about 10 "positive colonies". After I cut them with resdtriction enzymes, I always got shorter inserts that had different sizes in length. Hence, I think the competent cells might attack the insert at random manner. Additional cutting site within insert seemd not the reason. Because if there is additional cutting sites, I think the size of fragemnt should be the same. Does everybody think so? Please reply me if you have invaluable opinion.
Thnaks!