Hi all,
I am new here and I am about six weeks in to my first ever research project! (I just came here to kick-start my career as a researcher.) :)
The aim of my research project:
"The aim of this project is to clone two glial transcription factors, SOX9 and NFIA, from one plasmid into a baculovirus transfer vector. This will involve the separation of the transcription factor cassettes by restriction enzyme digestion. Once this is performed, construction of the recombinant baculovirus that expresses the mammalian glial reprogramming factors will take place. Polymerase chain reaction (PCR) will be utilised to confirm the correct construction of the transfer vector and recombinant baculovirus. If successful, the recombinant baculovirus may be used to induce expression in mouse embryonic stem cells or induced pluripotent stem (iPS) cells. If time permits, testing the key features that characterise astrocytes will confirm or falsify successful differentiation."
So far, over the past six weeks, myself and my supervisor have decided upon the following:
Plasmids from which we want to clone:
- tetO.Nfib.Hygro (https://www.addgene.org/117271/)
- tetO.Sox9.Puro (https://www.addgene.org/117269/)
Baculovirus into which we want to clone the transcription factors:
- pEZT-BM (https://www.addgene.org/74099/)
In an ideal world, we would splice with the following restriction enzymes:
NFIB
- 5′ cloning siteEcoRI (not destroyed)
- 3′ cloning siteBamHI (not destroyed)
SOX9
- 5′ cloning siteEcoRI (not destroyed)
- 3′ cloning siteXbaI (not destroyed)
However, the recipient plasmid available, pEZT-BM, does not have all of the required RE sites in the correct location; thus, the gene may not be placed in the correct orientation. We need to clone in the ORF using the sites available. If not, then we may need to do a "roundabout" , non-conventional, troubleshooting method. This is my first time doing gene cloning and stem cell research — a bit intimidating!
I have attached images of the plasmids we are using.
For further information, I have listed the following RE sites:
Sox9 (ORF = 17–2203):
XhoI = 1598, 2846, 10150 (will cut in the reading frame -> not useful)
NotI = 9075
KpnI = 2968, 10457
pEZT-BM:
XhoI = 6245
NotI = 6252
KpnI = 6279
We may, therefore, use NotI (forward) and KpnI (reverse) primers.
NfiB (ORF = 2824–4086 and up to 5704 if including IRES HYGRO):
NotI = 1294
KpnI = 2676, 3444, 4077, 4529, 6464 (will cut in the reading frame -> not useful)
NheI = 5708
BmtI = 5712
pEZT-BM:
NotI = 6252
KpnI = 6279
NheI = 6239
BmtI = 6249
Really struggling to find compatible RE here (the orientation does not seem to match if we use NotI and NheI).
We are also using PCR cloning to isolate the said genes once cut. I am a little hazy on this technique. Could I please have a little assistance in understanding this process?
Thank you and warm regards,
Joshua Nicholls
I would say that Gibson assembly will do a good job. see below,
https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly
In addition to the previous answer, it is important to know that you can use (1) a restriction enzyme that has only one restriction site at the site where you want to place your insert or (2) two different restriction enzymes that possess only one restriction site each along the inerting site of interest and in this case, few sequences that are in between the two sites will be replaced by the insert of interest.
Thank you, Abubakar Yakubu Saddeeq and Junjie Shao . I have just spoken to my supervisor to get his opinion. Unfortunately, due to time limitations and COVID-19-related restrictions, it will not be feasible for us to use Gibson assembly. We will simply be using traditional RE cutting, PCR isolation, and ligation.
As of now, I am struggling to find compatible REs between NFIB and the recipient, pEZT-BM.
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