Difference between revisions of "Team:SPSingapore/Notebook-Week-17"

Line 668: Line 668:
 
<span class = "nbcontent">
 
<span class = "nbcontent">
 
PCR 12 tubes, gradient (58-62degrees) for 30cycles
 
PCR 12 tubes, gradient (58-62degrees) for 30cycles
−
<br> Ran gel first three tubes had PCR product with large bands-gel extract
+
<br> Ran gel first three tubes had PCR product (Junk-pNirB-inv-suffix-Junk) with large bands-gel extract
 
</span><br>
 
</span><br>
  
 
<table class = "nbtable" style = "margin-left:50px;">
 
<table class = "nbtable" style = "margin-left:50px;">
−
<tr><td colspan = 2><b> RE of above PCR product (Junk-pNirB-inv-suffix-Junk) with EcoRI/PstI </b> </td></tr>
+
<tr><td colspan = 2><b> RE of above PCR product with EcoRI/PstI </b> </td></tr>
 
<tr><td> DNA </td><td> 50 </td></tr>
 
<tr><td> DNA </td><td> 50 </td></tr>
 
<tr><td> Buffer </td><td> 5 </td></tr>
 
<tr><td> Buffer </td><td> 5 </td></tr>
Line 842: Line 842:
 
<!----------------------new day------------------------------>
 
<!----------------------new day------------------------------>
  
 +
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; Sep 16
 +
<br><br>
 +
<span class = "brown"> Anaerobic Promoter</span>&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Inoculated colonies 12, 13, 19 of pSB1C3 placgfp and pAC placgfp, BL21 in 3mL LB at 7 am and 8am
 +
</span><br><br>
 +
<span class = "nbcontent">
 +
Colony PCR for pNirBinv in pSB1C3 using Biobricks suffix/prefix primers
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
 +
<tr><td> H2O </td><td> 22.75 </td></tr>
 +
<tr><td> PCR Buffer </td><td> 35 </td></tr>
 +
<tr><td> dNTP </td><td> 7 </td></tr>
 +
<tr><td> Primer </td><td> 7/7 </td></tr>
 +
<tr><td> MgCl2 </td><td> 17.5 </td></tr>
 +
<tr><td> Taq </td><td> 1.75 </td></tr>
 +
</table>
 +
<br>
 +
<span>
 +
&#9654; PCR was run for 24 cycles at Annealing temperature of 54, using the ‘COLONY protocol’
 +
<br> &#9654; Faint band of correct size was produced for colony 4
 +
</span>
 +
 +
 +
</div>
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
  
  
 
<!----------------------new day------------------------------>
 
<!----------------------new day------------------------------>
 +
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; Sep 17
 +
<br><br>
 +
<span class = "brown"> Anaerobic Promoter</span>&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Colony PCR for colony 4 of pSB1C3 was conducted using:
 +
<br> &emsp;&emsp;&emsp;&emsp; 1. VF/VR primers for Ta of 55
 +
<br> &emsp;&emsp;&emsp;&emsp; 2. BiobricksPrefix/Suffix primers for Ta of 57
 +
<br> &emsp;&emsp;&emsp;&emsp; 3. invlloF1/invendBBsuffixlonger for Ta of 65
 +
<br> with separate negative controls for each
 +
<br> &#9654; A band of the correct size, 2.8kb was produced for 2. and 3.
 +
</span>
 +
 +
</div>
 +
<br><br>
 +
 +
 +
<span class = "brown"> Anaerobic Promoter</span>&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Chi Yan &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
PCR of placgfppSB1C3 colony 12, JUnk prefix-placgfp-suffix-junk, placgfppAC, negative control of water
 +
</span><br>
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td colspan = 2><b> Mastermix </b> </td></tr>
 +
<tr><td> Buffer </td><td> 20 </td></tr>
 +
<tr><td> MgCl2 </td><td> 16 </td></tr>
 +
<tr><td> dNTPs </td><td> 4 </td></tr>
 +
<tr><td> Primers </td><td> 4/4 </td></tr>
 +
<tr><td> Taq </td><td> 1 </td></tr>
 +
</table>
 +
<br><br>
 +
 +
<span class = "nbcontent">
 +
PCR of same templates as above
 +
<br> &#9654; Primers: F2’_Prefixplac/RP_VR
 +
<br> &#9654; Same master mix as above
 +
</span><br>
 +
<span class = "nbcontent">
 +
Ran gel for above PCRs
 +
</span>
 +
 +
</div>
 +
<br><br>
 +
 +
 +
<span class = "brown"> Anaerobic Promoter</span>&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Mammalian cell invasion assay with BL21 and BL21+pSB1C3-pNirB-invasin under aerobic and anaerobic conditions
 +
</span>
 +
 +
</div>
 +
<br><br>
 +
 +
 +
<span class = "brown"> Anaerobic Promoter</span>&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Duy &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Confocal microscopy to determine relative levels of GFP fluorescence
 +
</span>
 +
 +
</div>
 +
<br><br>
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
 +
 +
  
  

Revision as of 13:00, 18 September 2015


Research Notebook

Week 12 (13/9 - 18/9)

▪ Sep 13

Invasin + Listerolysin   Anaerobic Promoter
☀ Chi Yan ☀

Ran gel for placgfp-psB1C3 colonies from Chi Yan 12/9 11pm. 1% 110V 30min
▶ no bands
▶ no positive colonies

Ran gel for PCRs to make Prefix-pNirB-inv-Suffix from Chi Yan 12/9 11pm
▶ E: successful, cut bands.
▶ F, G, H,I no bands

PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2

8 cycles 50-60degrees 2 min for annealing step
30 cycles 55-65 degrees 40s for annealing step

50ul, 3 tubes, put in columns 4, 8 12. Negative control added.
Buffer 15
dNTPs 6
MgCl2 6
Primers 1.5/1.5
Template 15
H2O 104.25
Taq 0.75


Invasin + Listerolysin   Anaerobic Promoter
☀ Yi Han ☀

Ran gel for YT PCR

PCR using F2 to produce F2/invendsuffix
Primer pairs are invlloF3new/invendsuffix and invlloF6/invendsuffix

PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2
invlloF3new/invendsuffix
Template (1000ng) 18
Primer 2/2
Buffer 20
sNTP 8
Taq 1
H2O 149
Total 200

invlloF6/invendsuffix
Template 500ng 9
Primer 1/1
Buffer 10
dNTP 4
Taq 0.5
H2O 74.5
Total 100


Attempt to make more of inv fragment from Yun Ting’s gel extract
Primers are FP2’/RP
Template 8.2
Primer 1/1
Buffer 10
dNTP 4
Taw 0.5
H2O 75.3
Total 100


Invasin + Listerolysin   Anaerobic Promoter
☀ Chi Yan & Yi Han ☀

PCR of invlloF1/lloendBBSuffix R with invasin D plasmid as template
▶ 5 cycles gradient 40-50degrees
▶ 20 cycles 62 degrees

Buffer 40
MgCl2 16
dNTPs 16
Primers 4/4
Template 9.2
H2O 308.8
Taq 2
Total 400

▶ 12 tubes at each temp, 33ul each

For YT: PCR of RP2/FP2’ using gel extract 1.6

Buffer 20
dNTP 8
MgCl2 8
Primers 2/2
Taq 1
H2O 135
Total 200
(3 tubes + 5ul template, 1 tube control)


Gel extract F1 invlloendsuffix
PCR of invlloend/BBsuffixR with invD
PCR of invlloF2/lloendBBsuffixR with F1/invlloendsuffix

F1invlloendsuffix
BUffer 80
MgCl2 32
dNTPs 32
Primers 8/8
Template 18.4
H2O 617.6
Taq 4
Total 800

Buffer 20
MgCl2 8
dNTPs 8
Primers 2/2
Template 84
H2O 75
Taq 1
Total 200

▶ 5 cycles 44-48 degrees
   25 cycles 60degrees

Colony PCR new placgfp-pSB1C3 following CY 12/9 11pm
Primers FP_KpnI_GFP/GFP_BBSuffix_new
60 degrees 25 cycles


▪ Sep 14

Anaerobic Promoter   ☀ Yi Han ☀

Ran gel for Chi Yan 13/9 9pm and 11pm
▶ Colony PCR no bands
▶ B1-12 no bands
▶ A1-16 bright band , correct size, extract


Anaerobic Promoter   ☀ Chi Yan ☀

PCR of invlloF1/lloendBBsuffixR with invD

Buffer 160
MgCl2 32
dNTPs 32
Primers 32/32
Template 18.3
H2O 517.7
Taq 8
Total 800

PCR of invloF2/lloendBBsuffix R with invlloF1endsuffixR
Buffer 80
MgCl2 16
dNTPs 16
Primers 16/16
Template 100
H2O 152
Taq 4
Total 400

Cycle conditions as of Chi Yan 13/9
Positions of tubes, A-C B-D
PCR done with GoTaq kit


Anaerobic Promoter   ☀ Yi Han ☀

Screen placgfp colonies for gfp using microscope
▶ Colonies 1, 4, 8 look promising

Miniprepped placgfp colonies
RE of colony 1, 4, 8
Template 1
EcoRI/PstI 0.1/0.1
Buffer 1
H2O 8
Total 10.2


Anaerobic Promoter   ☀ Chi Yan ☀

Sent colonies 1, 4, 8 of placgfppSB1C3 for sequencing with FP_BB_Prefix, RP_BB_Suffix, VF2.
Ran gel for YH placgfp pSB1C3 colonies RE 1, 4, 8
Incomplete digestion as only digested for 1.5hours
PCR of placgfp pSB1C3 colonies 1, 4, 8 from plasmids and with placfp pAC with FP_BB_Prefix and RP_BB_Suffix
Annealing temp 55degrees for 30s, extension for 60 seconds, for 25 cycles
GOTaq Master Mix 1
Buffer 20
MgCl2 10
dNTPs 4
Primers 4/4
Taq 1


Anaerobic Promoter   ☀ Yi Han ☀

PCR with GoTaq kit to make Fragment 8 from Fragment 1
Template (500ng) 40
dNTPs 4
MgCl2 16
Primers 2/2
GoTaq 0.5
PCR Buffer 20
H2O 19.5
Total 104.5


Continued from previous
Ran gradient at annealing temperature for 55-65, in wells 1, 4, 7, 12

Ran gel, no bands in all
    1. Made more F1 invsuffixendloger from invD
    2. Use BB PrefixpBirBinvstart/invendBBSuffixend to generate whole fragment
    3. Make fragment 8 using F8/invendsuffixlonger

Set PCR machine to 10 cycles at Ta gradient 40-55 degrees
30 cycles Ta 60 degrees
Ramp of 10%

PCR mix for 1 and 2
InvD Template 9.2
dNTPs 20
MgCl2 16
Primers 4/4
GoTaq 0.5
H2O 105.8

PCR mix for 3
Template 45
dNTP 8
MgCl2 8
Primers 1/1
GoTaq 1
PCR Buffer 20
H2O 16
Total 100

▶ Result: 1, 2 had clear bands, 3 had no bands


Anaerobic Promoter   ☀ Chi Yan ☀

PCR of placgfp colonies 1, 4, and 8 plasmids and placgfp pAC colony plasmid using VF2/VR
Annealing temperature is 52degrees for 30seconds, extension of 60s 30 cycles using GoTaq kit
Buffer 20
MgCl2 16
dNTPs 4
Primers 4/4
Taq 1

▶ Colony 1: Template 0.4/12.35 H2O
▶ Colony 4: Template 0.8/11.85 H2O
▶ Colony 8: Template 0.7/12.05 H2O
▶ pAC: Template 2.5/10.25


▪ Sep 15

esa Quorum Sensing   ☀ Adrian ☀

BBa_B0015 RE D
Buffer 4
H2O 6
EcoRI-HF 1
PstI-HF 1
Template 10
Total 20

Colony pcr for two samples
H2O 13.5
Buffer 5
MgCl2 2
dNTP 2
FP_BBPrefix_Junk 1
RP_BBPrefix_Junk 1
Taq 0.5
Total 25

Rapid DNA Ligation: 200ng insert: 21.4 ng vector


Anaerobic Promoter   ☀ Yi Han ☀

PCR with prefix-pNirB ultramer was successful.
PCR with Junk primers to add sequence
dNTPS 20
MgCl2 16
Primers 4/4
GoTaq 2
Buffer 40
Template (1500ng) 88
H2O 26
Total 200


PCR 12 tubes, gradient (58-62degrees) for 30cycles
Ran gel first three tubes had PCR product (Junk-pNirB-inv-suffix-Junk) with large bands-gel extract
RE of above PCR product with EcoRI/PstI
DNA 50
Buffer 5
EcoRI 0.5
PstI 0.5
Total 55


esa Quorum Sensing   ☀ Kenneth ☀

GFP Thingy
Buffer 4 2
H2O 6
EcoRI-HF 1
PstI-HF 1
Template 10
Total 20


Anaerobic Promoter   ☀ Chi Yan ☀

Colony PCR (and inoculation in 3mL LB+chl) of psB1C3-placgfp colonies 1, 4, 8, 11-31, placgfppAC, water. 15ul reaction *26 = 650
Mastermix
Buffer 130
MgCl2 104
dNTPs 26
Primers 26/26
Taq 6.5
(H2O 11.75 each)
(12.25ul mastermix each tube)

Run gel for YH 15/9 RE of pSB1C3 (gel purified) and Junk-pref-pNirB-inv-suffix-junk with EcoRI/PstI


Anaerobic Promoter   ☀ Yi Han ☀

Extract of the RE digested BB-Prefix-pirB-inv-suffix

Ligation Reaction
Template (insert) 60
Ligase 3
10X buffer 3
Cut pSB13 1.35


PCR to get more Junk-prefix-pNirB-Suffix-junk
Template (2550ng) 150
dNTPs 33
MgCl2 25.6
Primer 6.4/6.4
GoTaq 3.2
Buffer 64
Total 250


Anaerobic Promoter   ☀ Chi Yan ☀

Run gel for CY 15/9 4pm colony pcr
▶ 1kb, 100bp, 1, 4, 8, 11-23, 100bp, 1kb
▶ 1kb, 100bp, 24-31, PCR Junk-Prefix-placgfp-suffix-Junk, pSC, H2O, 100bp, 1kb

Positive result with colony PCR for colonies 12, 13, 15, 17 (does not glow), 19, 20
▶ Duy used confocal microscopy to check for GFP expression
▶ Sequencing results showed that colonies 12, 13 and 19 has correct sequence.


Anaerobic Promoter   ☀ Chi Yan ☀

gel extract Junk-prefix-pNirB-inv-suffix-junk from YH 15/9 7pm
RE with EcoRI/PstI 2hours at 37 degrees
DNA 30
Buffer 3
EcoRI 0.5
PstI 0.5


The ligated plasmid was then transformed into 10ul BL21.
7:1 ligation reaction
Insert 10.4
Vector 1.5
Ligase 1
Buffer 2
Water 5.1

5:1 ligation reaction
Insert 7.56
Vector 1.5
Ligase 1
Buffer 2
Water 8.04


▪ Sep 16

Anaerobic Promoter   ☀ Yi Han ☀

Inoculated colonies 12, 13, 19 of pSB1C3 placgfp and pAC placgfp, BL21 in 3mL LB at 7 am and 8am

Colony PCR for pNirBinv in pSB1C3 using Biobricks suffix/prefix primers
H2O 22.75
PCR Buffer 35
dNTP 7
Primer 7/7
MgCl2 17.5
Taq 1.75

▶ PCR was run for 24 cycles at Annealing temperature of 54, using the ‘COLONY protocol’
▶ Faint band of correct size was produced for colony 4


▪ Sep 17

Anaerobic Promoter   ☀ Yi Han ☀

Colony PCR for colony 4 of pSB1C3 was conducted using:
     1. VF/VR primers for Ta of 55
     2. BiobricksPrefix/Suffix primers for Ta of 57
     3. invlloF1/invendBBsuffixlonger for Ta of 65
with separate negative controls for each
▶ A band of the correct size, 2.8kb was produced for 2. and 3.


Anaerobic Promoter   ☀ Chi Yan ☀

PCR of placgfppSB1C3 colony 12, JUnk prefix-placgfp-suffix-junk, placgfppAC, negative control of water
Mastermix
Buffer 20
MgCl2 16
dNTPs 4
Primers 4/4
Taq 1


PCR of same templates as above
▶ Primers: F2’_Prefixplac/RP_VR
▶ Same master mix as above
Ran gel for above PCRs


Anaerobic Promoter   ☀ Yi Han ☀

Mammalian cell invasion assay with BL21 and BL21+pSB1C3-pNirB-invasin under aerobic and anaerobic conditions


Anaerobic Promoter   ☀ Duy ☀

Confocal microscopy to determine relative levels of GFP fluorescence