#  sgRNA cloning and sequencing 

 



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  expand\_more  

 
  

 

 Cloning sgRNAs Sequencing Inserts 

## Cloning sgRNAs

 

 

pCFD3 - The TRiP uses the protocol described at [crisprflydesign](http://www.crisprflydesign.org/plasmids/) - [**PDF**](/file_url/546)

pCFD4 - The TRiP uses a modified version of the protocol from [crisprflydesign](http://www.crisprflydesign.org/plasmids/) - [**PDF**](/file_url/545)

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

 ![pcfd4-ms2_primers](/sites/g/files/omnuum5366/files/2024-09/pcfd4-ms2_primers.jpg)

 

pl100 - The protocol is adapted from Housden et al, 2014 - [**PDF**](/file_url/548)



 



 

 

 

## Sequencing Inserts

 

 

### Genotyping TRiP-OE or TRiP-KO stocks for the presence of pCFD4 and pCFD3 insertions **[PDF](/file_url/556)**


### 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

Sort2X 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

SortStep1:

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

SortStep1:

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: GTC**G** + sgRNA + GTTTTAGAGC  
**b.** for pCFD4 the sequence is: AACTTC + sgRNA1 + 496bp + sgRNA2 + GTTTT