Difference between revisions of "Team:SPSingapore/Notebook-Week-9"

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<br> Expected is 1.5+0.1 kb (from VR).  
 
<br> Expected is 1.5+0.1 kb (from VR).  
 
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Colony PCR for inv/hly plasmid using Biobricks Prefix and Suffix primers, and Universal primers VF, M2 (Using gradient PCR and different annealing temperature) for 5 samples
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Ran gel in same order with 1kb, 100bp followed
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<span class = "brown"> Anaerobic Promoter</span>
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&emsp;&emsp;
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<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Chi Yan &#9728;</font>
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Ligation of Cut FNRGFP (17/7) with cut GFP vector
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<tr><td colspan = 2> 1X reaction </td></tr>
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<tr><td> Buffer </td><td> 2ul </td></tr>
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<tr><td> 200ng vector </td><td> 1.1ul </td></tr>
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<tr><td> 3:1 </td><td> 256.5ng </td></tr>
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<tr><td> Water </td><td> 3.4ul </td></tr>
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<tr><td> T4 DNA ligase </td><td> 1ul </td></tr>
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<tr><td> Total </td><td> 20ul </td></tr>
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&#9654; Incubation at room temperature for 2h
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<br> &#9654; Transformation into 10ul BL21 competent cells
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Revision as of 21:07, 17 September 2015


Research Notebook

Week 9 (19/7 - 25/7)

▪ July 19

Invasin + Listerolysin    ☀ Yi Han ☀

Inoculation of positive colonies in liquid culture. 3mL and shaking incubation.
inoculated colonies C,D for invasin plasmid in 3mL LB+amp at 1.30pm
Transformed remaining 40u ligation reaction for FNR gfp into 10ul BL21
Transformed 100ng pGFPuv plasmid into 10ul BL21
Poured new LB+amp plates
Plated 20, 40, 100ul of transformed bacteria in SOC media on plates


esa Quorum Sensing    ☀ Clarice ☀

Colony PCR for RNG and esaR-GFP colonies from master plate
RNG (10X) esaGFP (9X)
H2O 13.8 45
Buffer 50 18
MgCl2 20 18
dNTPs 20 9
Primer 1 10 9
Primer 2 10 9
Gotaq 2 1.8


▪ July 20

Invasin + Listerolysin    ☀ Yun Ting ☀

Miniprep of positive colonies from 30h liquid culture.
Eluted in 50ul elution buffer and stored at -20.
▶ C= 225.9ng/ul. 260/280=1.86
▶ D= 297.7ng/ul. 1.87


Anaerobic Promoter    ☀ Yi Han ☀

1. pGFPuv transformation worked!
2. Single colony for FNR-GFP + 4 more from plating on 19/7
3. Sent FNR-GFP fusion product for sequencing with RP-Biobricks Suffx - result there is no FNR but there is no GFP
4. Colony PCR for FNR gfp (Colonies 1-5, (-) control)

Colony PCR (6X)
H2O 19.5ul
Buffer 10ul
dNTP 6ul
FP_Biobricks Suffix 6ul
RP_Biobricks SUffix 6ul
MgCl2 15ul
Taq Polymerase 1.5ul


▪ July 21

Invasin + Listerolysin    ☀ Yun Ting ☀

Colony PCR with thermogradient
H2O 70ul
dNTP 14ul
MgCl2 35ul
Taq 3.25ul
primers (forward + reverse) 14*2
Template DNA 14


C/D-1,2 Col3.
C/D-3,4 Col12.
D-5,6 Col 9
D-7,8 Col 10
Negative control (no template) Col 8

▶ Gel ran for 80V, 1h.
     D-1,2: VF2, VR.
     D-3,4: FP-prefix, RP-suffix.
     D-5,6: FP-prefix, FP_M2.
     D-7,8: FP-prefix, FP_invF.
     C-1,2: VF2, VR.
     C-3,4: FP-prefix, RP-suffix.

▶ Results Notes: Too much template DNA (~250ng). Separate the universal primers (to avoid differing extension time).


Anaerobic Promoter    ☀ Chi Yan ☀

Ran gel for 20/7 colony pcr for FNR gfp
1kb, 100bp, colonies negative control


Anaerobic Promoter    ☀ Yi Han ☀

Redid colony pcr for above as negative control had a positive band - contamination of PCR
negative control still has band - reagents contaminated


▪ July 22

Anaerobic Promoter    ☀ Yi Han ☀

Miniprepped colonies 1-5 for FNR-gfp plasmid

Did EcoRI/PstI digest
Digest for mastermix (5X)
Buffer 10ul
EcoRI/PstI 2ul
H2O 60ul
Total 20ul per tube
(400ng plasmid in each tube)

Ran gel for RE samples, none were positive clones


Anaerobic Promoter    ☀ Chi Yan ☀

PCR to test ‘FNR-GFP plasmid’. Colonies 1-5 from 20/7 + negative control
3 sets of primers
   1. FP_BB Prefix, RP BB Suffix
   2. GFP-F, GFP-R
   3. FP_FNRPromoter_BBP, ORP_FNR_Promoter GFP

For each reaction (6X)
H2O 73.5ul
dNTPs 6ul
Forward/Reverse Primer 6ul/6ul
MgCl2 15ul
Taq polymerase 1.5ul
Plasmid 1ul
Buffer 30ul


▪ July 23

Invasin + Listerolysin    ☀ Yan Ting ☀

RE digest of inv/hyl plasmid with EcoRI and PstI for 2 hours at 37oC.
▶ Lane 1-4: 1kb ladder, 100bp ladder, RE of colony C, RE of colony D.
▶ Size is correct: 4kb main band (inv+hly part) and 2kb (plasmid backbone). These plasmid DNA should have the part.


▪ July 24

Invasin + Listerolysin    ☀ Yun Ting & Yan Ting ☀

Colony PCR, used with temperature gradient to vary annealing temperature.
H2O 127.5
5x buffer 50
dNTP 10
MgCl2 25
Taq 2.5
FP + RP 10*2
Template DNA 75ng per rxn
100V for 45min (can run for longer).

Lane 1: 1kb ladder; lane 2: 100bp ladder;
lane 3-7: FP prefix + RP suffix (5 repeats with increasing annealing temperatures) has 2 bands ~800 bp & 100-200bp (probably non specific bands, will lower # of cycles in future, use higher annealing temp, lower annealing time).
Expected size of inv+hly part is 4.1kb.
lane 8-9: VF + M2 has a thick band 800-900bp.
Expected size is 600+130bp (VF adds ~130bp compared to FP_prefix) .: doesn’t seem to be correct….
lane 10-11: VF + inv F has no bands. Expected is at least 200bp (bcos universal primers).
lane 12: VR + inv F seems to have a small band <100bp. Non specific amplification?
Expected is 1.5+0.1 kb (from VR).

Colony PCR for inv/hly plasmid using Biobricks Prefix and Suffix primers, and Universal primers VF, M2 (Using gradient PCR and different annealing temperature) for 5 samples
Ran gel in same order with 1kb, 100bp followed


Anaerobic Promoter    ☀ Chi Yan ☀

Ligation of Cut FNRGFP (17/7) with cut GFP vector
1X reaction
Buffer 2ul
200ng vector 1.1ul
3:1 256.5ng
Water 3.4ul
T4 DNA ligase 1ul
Total 20ul
▶ Incubation at room temperature for 2h
▶ Transformation into 10ul BL21 competent cells