sgRNA cloning and sequencing

pCFD3 - The TRiP uses the protocol described at crisprflydesign - PDF

 

pCFD4 - The TRiP uses a modified version of the protocol from crisprflydesign - PDF

 

pCFD4-MS2 - The protocol is the same as for pCFD4, except for the primer sequence
 

pCFD4-MS2 primers

 

pl100 - The protocol is adapted from Housden et al, 2014 - PDF

 

Genotyping TRiP-OE or TRiP-KO stocks for the presence of pCFD4 and pCFD3 insertions PDF

 

Prepared by Rong Tao and Jonathan Zirin

 

For TRiP-OE stocks in pCFD4, the primers are:

pCFD4-F: 5'-GACACAGCGCGTACGTCCTTCG-3'
pCFD4-R: 5'-ACTCTCAGGCTCCAGGTAGG-3'

For TRiP-KO stocks in pCFD3, the primers are:

pCFD3-F: 5'-ACGTTTTATAACTTATGCCCCTAAG-3'
pCFD3-R: 5'-GCCGAGCACAATTGTCTAGAATGC-3'

1. Crude genomic DNA preparation:
SB buffer:
10mM Tris-HCl pH8
1mM EDTA
25mM NaCl
Proteinase K: Stock conc. (100X): 20mg/ml
-Working conc.: 200ug/ml (actually 20ug/ml works fine)
-add Proteinase K to SB buffer just before experiment

a. place a single fly in an Eppendorf tube
b. add 50ul SB buffer
c. homogenize
d. incubate at 37°C ´ 30min
e. spin: 16,000g ´ 2 min
f. transfer the supernatant to PCR tube
g. incubate 95°C ´ 3min
h. store at 4°C (for longer term, store in -20°C freezer)

2. Genotyping PCR:
Use these primers for pCFD4 to amplify a band of 731bp
pCFD4-F: 5’-GACACAGCGCGTACGTCCTTCG-3’
pCFD4-R: 5’-ACTCTCAGGCTCCAGGTAGG-3’

Use these primers for pCFD3 to amplify a band of ~500bp
pCFD3-F: 5’-ACGTTTTATAACTTATGCCCCTAAG-3’
pCFD3-R: 5’-GCCGAGCACAATTGTCTAGAATGC-3’

a. PCR rxn

2X GoTaq Green15 ul
H2O11.25 ul
FW primer (10uM)0.625 ul
RV primer (10uM)0.625 ul
Genomic DNA 2.5 ul

b. PCR program for pCFD3

Step1:95°C, 2 min
Step2(35 x):95°C, 30 sec
50°C, 30 sec
72°C, 1 min
Step3: 72°C, 10 min
Step4: 

4°C

        

 

 

 

 

 

c. PCR program for pCFD4

Step1:95°C, 2 min
Step2(35 x):95°C, 30 sec
55°C, 30 sec
72°C, 1 min
Step3:72°C, 10 min
Step4: 4°C

 

 

 

 

 

              

3. Run gel:

a.run 4 ul PCR product on agarose gel
b.check if PCR produces the right size of band and enough DNA for doing PCR purification

4.PCR purification:

a.use QIAquick PCR Purification Kit
b.elute in ~50 ul H2O
c.purified DNA should be 10~20ng/ul

5. Sequence purified PCR product:

a. for pCFD3 use pCFD3-F primer
b. for pCFD4 use pCFD4-F primer

6.Sequencing data analysis:

a. for pCFD3 the sequence is: GTCG + sgRNA + GTTTTAGAGC
b. for pCFD4 the sequence is: AACTTC + sgRNA1 + 496bp + sgRNA2 + GTTTT