TITLE: Cloning into pSL2680 CRISPR Plasmid - iGEM IISER Pune 2021
AUTHORS: misaal.bedi 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to clone the guide RNA and Homologous Repair Template into the pSL2680 plasmid which was a gift from Himadri Pakrasi (Addgene plasmid # 85581 ; http://n2t.net/addgene:85581 ; RRID:Addgene_85581).</div></div>

[STEPS]
?. [Cloning the gRNA]
The gRNA is to be cloned before the cassette into the plasmid.
?. [Day 1]
Take  of LB media with   Kanamycin and scratch off some solid culture from the vial and add to the media in a flask. Grow this   at
30 mL
37 °C
?. [Day 2]
Perform miniprep on the entire  culture (since the pSL2680 plasmid is of a low copy number). Spin it down and resuspend it in  resuspension buffer in the fridge. Divide it into two sets and follow the rest of the miniprep protocol. In the end, elute from the column with   of distilled water.
30 mL
500 µl
50 µl
?. [Day 2]
Digest the  of miniprep with AarI using the following reaction mix:AarI buffer - plasmid prep - 50X AarI oligo - Aarl enzyme - Distilled water - Incubate at   for 4 hours.
50 µl
10 µl
50 µl
2 µl
4 µl
34 µl
37 °C
?. [Day 2]
Gel extract the digest. Run the entire digest in a double or triple lane on a 0.7-1% agarose gel. Excise the largest band and place it in a  Eppendorf tube. Melt at   (Tip: Inverting the tube every five minutes helps in faster melting)
2 mL
55 °C
?. [Day 2]
Perform gel purification using a gel purification kit and follow its protocol.
?. [Day 2]
In the end, elute with  of water (ensure that a small amount of water is placed in the center of the membrane).
20 µl
?. [Day 3]
Anneal the gRNA oligos with the following reaction mixture: stock of the gRNA left primer -  stock of the gRNA right primer - Ligase buffer - Distilled water - PNK enzyme - Use the following thermocycler program:Heat to   for 5 minutes, and then ramp to   at  /second.
10 µl
10 µl
5 µl
25 µl
1 µl
95 °C
4 °C
0.1 °C
?. [Day 3]
Dilute the annealed oligos 1:50. Not a lot is required so  with  of water can be used.Ligate the oligos into pSL2680 which was digested the previous day with the following reaction mix:  pSL2680 gel extract - Annealed oligos that were dilute 1:50 - Ligase buffer - Ligase - Incubate  at
2 µl
98 µl
8 µl
0.5 µl
1 µl
0.5 µl
16 °C
?. [Day 4]
Transform the entire  ligation reaction into the E. coli XL1 - blue or Top10 strains as follows:Thaw cells  for 10 minutes.Add  ligation reaction to cells and stir. Don't pipette up and down.Incubate  for another 10 minutes.Heat shock for 45 seconds at  .Place  for 2 minutes.Add  of liquid LB media.Incubate at  for 1 hour.
10 µl
on ice
10 µl
on ice
42 °C
on ice
500 µl
37 °C
?. [Day 4]
Centrifuge the reaction mixture after incubation and remove the supernatant. Add the pellet to  of the media. Plate transformants on LB media with   Kanamycin and add IPTG+X-gal.Spread   of   IPTG and    X-gal on a plate with LB media with   Kanamycin and let it soak in for at least an hour.Prepare the plates before starting the transformation.
50 µl
40 µl
?. [Day 5]
Pick 8 white colonies from the transformants and patch them on LB media with   Kanamycin.Start  cultures of 2 colonies off the plate in LB media with   Kanamycin.
10 mL
?. [Day 6]
Isolate the plasmid from the 2 cultures incubated the previous day. Determine the concentration and send for sequencing. Once the correct sequence is confirmed, prepare stocks.
?. [Cloning of cassette ]
After the gRNA has been cloned into the plasmid, we can begin cloning the cassette to make modifications in the S. elongatus UTEX 2973 genome.
?. [Day 1]
Synthesize the repair template using high-fidelity PCR. Gel extract the PCR reactions and purify them, and elute in   of distilled water.Digest the plasmid carrying the gRNA using Kpnl and make sure to dephosphorylate it, to prevent recircularization of the backbone.Use the following reaction mixture:Buffer - Plasmid Prep - Kpnl restriction enzyme - FastAP (dephosphorylates vector) - Distilled Water - Incubate for 4 hours at Gel extract the digest and elute in   of distilled water.
25 µl
15 µl
75 µl
3 µl
53 µl
37 °C
25 µl
?. [Day 2]
Concentrate the gel extractions and perform Gibson Assembly and concentrate the DNA.Allow samples to dry for 2.5-3 hours.For Gibson Assembly, exactly  of correctly mixed and highly concentrated DNA is required. Use of the vector and a 2X molar ratio of each fragment to be assembled by Gibson Assembly. Note: Use molar ratios of the fragments, not weight.The vector and PCR fragments should be mixed using thePrepare the mixture in a PCR tube with a total volume of  . Add  of the Gibson master mix to the  of DNA. Mix them together and PCR at  for an hour and then hold at
5 µl
200 ng
5 µl
15 µl
5 µl
50 °C
4 °C
?. [Day 3]
Transform at least  of the Gibson reaction in XL1-blue (or Top10) using the same protocol as described in step 5 of Day 4 in the 'Cloning the gRNA' section of this protocol.Plate on LB media with   Kanamycin
10 µl
?. [Day 4]
Check the transformed colonies by PCR.Run the reactions on a gel and select two positive colonies. Start with   of each colony in LB media with   Kanamycin for plasmid isolation.
10 mL
?. [Day 5]
Isolate plasmids from the two colonies isolated the previous day and elute in  .Sequence the insert in the plasmid. If the sequencing is correct, this protocol has been successfully completed and all that remains is to transform the plasmid into cyanobacteria.Note: Don't forget to freeze the recombinant E.coli in permanent stock.
50 µl