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.

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