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
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 Green | 15 ul |
H2O | 11.25 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