Hello -
I am studying this protocol which claims to use pcr to clone in/replace a small piece of an sgrna plasmid with a new guide sequence provided by an oligo with homology to the sgrna scaffold. Then one transforms e coli with the resultant pcr reaction. My question is : how does pcr not linearize the entire product? Here is the protocol :
2. Order two 60-mer oligonucleotides, one with the sgRNA guide coding sequence (Step 1) and one
with the reverse complement of the coding sequence.
3. Dilute each of the two 60-mers to 100 μM in water.
4. Assemble the reaction in a polymerase chain reaction (PCR) tube:
Phusion HF buffer (5×)
5 μL
dNTP (10 mM total; 2.5 mM each)
0.5 μL
Coding sgRNA guide sequence (60-mer; 100 μM)
0.1 μL
Reverse complement of sgRNA (60-mer; 100 μM)
0.1 μL
pCAS plasmid
40 ng
Phusion DNA Polymerase
1 μL
H2O
to 25 μL
5. Perform thermocycling with the following profile.
1 cycle
98 ̊C
1 min
30 cycles
98 ̊C
30 sec
58 ̊C
1 min
72 ̊C
10 min
1 cycle
72 ̊C
10 min
4 ̊C
Hold
6. Add 1 μL of DpnI, 3 μL of the provided 10× digestion buffer, and 1 μL of water to the cloning
reaction and incubate for 6 h at 37 ̊C (or overnight).
7. Transform the DpnI-treated reaction into E. coli competent cells:
i. Add 15 μL of the cloning reaction into 50 μL of ice-thawed competent bacterial cells.
ii. Incubate on ice for 30 min.
iii. Heat shock in a heat block or water bath for 1 min at 42 ̊C.
iv. Recover in 200 μL of LB liquid medium for 1 h at 37 ̊C.
v. Spread the contents of the reaction onto LB+ kanamycin plates.
vi. Incubate overnight at 37 ̊C.
8. Pick five to 10 colonies from the LB+ kanamycin plate and shake-incubate overnight in 3 mL of
LB+ kanamycin at 37 ̊C.
9. Isolate the plasmids using a QIAGEN Miniprep Kit and quantify DNA using the NanoDrop
spectrophotometer (1 A260 OD = 50 ng/μL).
10. Sequence the sgRNA construct by Sanger sequencing with the sequencing primer 5′-CGGAA
TAGGAACTTCAAAGCG-3′ .
attached is the entire paper
thanks!
- Amy J.
I have got this protocol to work, as far as getting the right band on a gel anyway. But I still don't know if I have linear dna or not....linear dna runs the same speed on a gel as a supercoiled plasmid with constructs close to 10 kb
This paper appears to answer my questions Article An efficient one-step site-directed deletion, insertion, sin...
some sage advice I received
"it uses long primers with say 30 bases of complementary sequence .If each primer faces away from the other it will read in a circle and extend through the other primer in each case. Linear. Then later as the reaction cools the ends will anneal and the linear template will become circular. If you have a unique RE site in the circle then cutting with that enzyme will cut the circle to one size that will run at a different place from the circle. If the product is already linear then you will produce 2 smaller linear fragments."
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