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Team:Freiburg/Notebook/lab crrna plasmid
From 2013.igem.org
Labbook RNA plasmid - pIG0021
As a central part of our toolkit we need to clone a plasmid that contains a tracrRNA and a locus for inserting CRISPR RNAs into the PSB1C3 backbone. The general strategy is to amplify both loci from the px334a plasmid by Feng Zhang and combine them in the iGEM backbone. iGEM biobrick cutting sites will allow us to combine several targets with no big effort.
August
28.08.13
All primers arrived, PCRs for fragments.
pU6:crRNA is amplified from px334a by PCR using the primers oIG0056 and the oU6:crRNA_fw primer. pH1:tracrRNA is amplified from pX334a by PCR suing the primers oH1:tracrRNA_fw and oH1:tracrRNA_rev.
PCR was performed using the following master mix and reaction conditions.
In the same reaction the backbone of PSB1C3 was amplified using the primers oIG0037 and oIG0038.
| µl | type |
|---|---|
| 10 | Q5-HF Reaction Buffer |
| 1 | Template |
| 1 | Primer1 |
| 1 | Primer2 |
| 4 | dNTPs |
| 1 | DMSO |
| 0.5 | Q5-HF Polymerase |
| Add to 50 | H2O |
- Annealing: 60°C
- Elongation: 120 sec
- 24 cycles
Expected sizes are ~500bp for the RNA fragments and 2kb for the backbone.
Somebody must have mixed up TAE and ddH2o by preparating agarose. The gel was not really resolving the DNA. Nevertheless, the fragments contained the approximate size fragments. Bands were cut out and extracted using the Roche GelEx kit.
Yields:
pH1:tracrRNA: 2.2ng/ul
pU6:crRNA: 5.6 ng/ul
backbone: 22.1 ng/ul
Fusion PCR with all three fragments
As earlier approaches showed these fragments are problematic when using multi-fragment Gibson cloning or classical cloning respectively. Therefore the fragments were ligated by fusion PCR.
| µl | type |
|---|---|
| 10 | Q5-HF Reaction Buffer |
| 3 | pH1:tracrRNA |
| 1 | PSB1C3 backbone |
| 3 | pU6:crRNA |
| 4 | dNTPs |
| 1 | DMSO |
| 0.5 | Q5-HF Polymerase |
| Add to 50 | H2O |
first PCR
- Annealing: 60°C
- Elongation: 180 sec
- 6 cycles
second PCR
1ul of oH1:tracrRNA_fw and oIG0038 were added for complete amplification of linearised RNA plasmid.
- Annealing: 60°C
- Elongation: 180 sec
- 24 cycles
This was done as a duplicate and put on a 1% agarose gel. The expected band runs at 3kb.
 |
| PCR aIG0021 |
Roth 1kb ladder was loaded. There are three bands visible, but only the most upper band fits the expected size. This band was cut out in both lanes and extracted by the Roche GelExtraction kit.
Yields
lane1: 9.6 ng/ul
lane2: 19.6 ng/ul
Gibson assembly for pIG0021
As the extracted linearised RNA plasmid contains complementary overhangs a 1-fragment Gibson was performed. To an aliquot of Gibson master mix 2.5 ul, respectively 5 ul of DNA was added and filled up with ddH2O to 5 ul. This mixture was incubated on 50° for 1 hour. Afterwards it was incubated for 3 minutes on ice and 3 minutes on RT.
5ul of this mixture was transformed into chemically competent E.coli and spread on chlorampheicol containing Agar plates.
29.08.2013
Colony PCR
Clones were obtained. Colony PCR was performed to screen for potential positive clones. 15 clones were picked, transferred to Master Mix, transferred to Master Plate.
| µl | type |
|---|---|
| 2.5 | standard Taq buffer |
| 1 | oIG6017 |
| 1 | oIG6018 |
| 2.5 | dNTPs |
| 0.125 | Taq Polymerase |
| Add to 25 | H2O |
- Annealing: 60°C
- Elongation: 80 sec
- 25 cycles
 |
| cPCR pIG0021 |
Clones with bands at roughly 1.2 kb bands seem to be positive, but a little bit to high. Nevertheless, those clones were streak out on plates for mini prep.
30.08.2013
Clones were prepped using the Promega mini prep kit. Test digest was performed using XbaI and Pst1-HF. Expected band sizes are 2kb and 0.9 kb.
| µl | Substance |
|---|---|
| 1 | Plasmid |
| 1 | CutSmart buffer |
| 0,5 | XbaI |
| 0,5 | Pst1-HF |
| 7 | H2O |
- Incubation: 1.5 h at 37° C
Results of test digest
 |
| 6 out of 8 colonies were positive. Clone 2 and 4 were sent for sequencing with oIG6017. |
September
04.09.2013
after trouble with the sequencing company results are finally available. Clone 2 is sequenced as correct. RNA plasmid is correct as as planned. For further usage 2 re-transformations were performed to gain enough material for further experiments.
11.09.2013
New plan: building multiple target RNA plasmids to avoid the additional cell stress, caused by multiple transfection with single target RNA plasmids. 1: VEGF target 4 + Bla target 2; 2: Bla target 3 + Bla target 4
Digest
| µl | type |
|---|---|
| 2 | VEGF target 2 plasmid / Bla target 3 plasmid |
| 5 | Cut Smart Buffer |
| 1 | Spe |
| 1 | Pst |
| Add to 50µl | H2O |
- Temp.: 37°C
- Incubation time: 2h
| µl | type |
|---|---|
| 2 | Bla target 2 / Bla target 4 |
| 5 | Cut Smart Buffer |
| 1 | Xba |
| 1 | Pst |
| Add to 50µl | H2O |
- Temp.: 37°C
- Incubation time: 2h
Gelex
Digest was load on gel; bands were cut out; Gelextraction
Ligation
| µl | type |
|---|---|
| 0,58 | digested VEGF 2 target |
| 16,67 | digested Bla target 2 |
| 1 | T4 DNA Ligase |
| 2 | Buffer |
| Add to 20µl | H2O |
- Incubation for 15 minutes (RT)
| µl | type |
|---|---|
| 1,15 | digested Bla target 3 |
| 12,82 | digested Bla target 4 |
| 1 | T4 DNA Ligase |
| 2 | Buffer |
| Add to 20µl | H2O |
- Incubation for 15 minutes (RT)
→ pIG4311, pIG4312 heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells
13.09.2013
Colony PCR
| µl | type |
|---|---|
| 2,5 | Taq Buffer |
| Template | |
| 1 | Primer1 (oIG6017) |
| 1 | Primer2(oIG6018) |
| 2.5 | dNTPs |
| 0.125 | Taq Polymerase |
| Add to 25 | H2O |
Sequencing of positive Clones
Sequences positive
15.09.2013
plan: multiple target RNA plasmid with VEGF target 4, Bla target 2, Bla target 3, Bla target 4
Digest
| µl | type |
|---|---|
| 2 | pIG4311 |
| 5 | Cut Smart Buffer |
| 1 | Spe |
| 1 | Pst |
| Add to 50µl | H2O |
| µl | type |
|---|---|
| 2 | pIG4312 |
| 5 | Cut Smart Buffer |
| 1 | Xba |
| 1 | Pst |
| Add to 50µl | H2O |
- Incubation for 15 minutes (RT)
| µl | type |
|---|---|
| 1,25 | digested pIG4311 |
| 3,63 | digested pIG4312 |
| 1 | T4 DNA Ligase |
| 2 | Buffer |
| Add to 20µl | H2O |
- Incubation for 15 minutes (RT)
→ pIG4313 heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells
13.09.2013
Colony PCR
| µl | type |
|---|---|
| 2,5 | Taq Buffer |
| Template | |
| 1 | Primer1(oIG6017) |
| 1 | Primer2(oIG6018) |
| 2.5 | dNTPs |
| 0.125 | Taq Polymerase |
| Add to 25 | H2O |
Sequencing of positive Clones
Sequence positive
